Phylogenetic analysis of the Argonaute protein family in platyhelminths

Argonaute proteins (AGOs) are mediators of gene silencing via recruitment of small regulatory RNAs to induce translational regression or degradation of targeted molecules. Platyhelminths have been reported to express microRNAs but the diversity of AGOs in the phylum has not been explored. Phylogenetic relationships of members of this protein family were studied using data from six platyhelminth genomes. Phylogenetic analysis showed that all cestode and trematode AGOs, along with some triclad planarian AGOs, were grouped into the Ago subfamily and its novel sister clade, here referred to as Cluster 1. These were very distant from Piwi and Class 3 subfamilies. By contrast, a number of planarian Piwi-like AGOs formed a novel sister clade to the Piwi subfamily. Extensive sequence searching revealed the presence of an additional locus for AGO2 in the cestode Echinococcus granulosus and exon expansion in this species and E. multilocularis. The current study suggests the absence of the Piwi subfamily and Class 3 AGOs in cestodes and trematodes and the Piwi-like AGO expansion in a free-living triclad planarian and the occurrence of exon expansion prior to or during the evolution of the most-recent common ancestor of the Echinococcus species studied.     

Identification and characterization of Piwi subfamily in insects

As a subfamily of Argonaute proteins, Piwi is poorly understood compared with Ago subfamily until recent discovery of Piwi protein interacting with piRNA. We did a large scale screening of insect genomes to identify piwi-like genes. Full or partial cDNA sequences were obtained by EST elongation and GENSCAN. We found that the exon numbers were totally different between vertebrates and invertebrates, approximately 20 exons in mammals but only 6-9 exons in insects. This infers either intron insertion or loss occurred during evolution. Characterized PAZ, c-terminal PIWI domains exist in almost all predicted Piwi-like proteins. We found six conserved motifs, which contain active catalytic triad "Asp-Asp-His/Lys" required for slicer activity. The expression of siwi1 and siwi2 in Bombyx mori were verified with RT-PCR. Phylogenetic tree inferred by Bayesian algorithm indicates invertebrate Piwi-like proteins are classified into three clades, of which Ago3 clade is closer to mammalian Piwi proteins.     

Identification,chromosomal mapping and conserved synteny of porcine Argonaute family of genes

The Argonaute proteins are recently identified and evolutionarily conserved family with two subfamilies Ago and Piwi, which play important roles in small RNA pathways. Most species have eight Argonaute members in their genomes, ranging from 1 to 27. Here we report identification of six Argonaute genes in pig, four members of the Ago subfamily (Ago1, Ago2, Ago3 and Ago4) and two members of the Piwi subfamily (Piwil1 and Piwil2), which were predicted to encode proteins of 857, 860, 860, 861, 861 and 985 amino acids, respectively. Phylogenetic analysis showed that the porcine Ago and Piwi genes were clustered into relevant branch of mammalian Argonaute members. The porcine Ago4- Ago1-Ago3 genes are linked together at the p12 of the chromosome 6, while Ago2 is located at the p15 of the chromosome 4. The porcine Piwil1 and Piwil2 are mapped together onto the chromosome 14, at the q14 and q11 respectively. Comparatively mapping of the Argonaute members on chromosomes showed that linkage group of the Ago4-Ago1-Ago3 and several neighborhood genes is evolutionarily conserved from chicken to mammals. The genes Piwil1 and Piwil2 are separated onto different chromosomes from fish to mammals, with exception to this tendency in both pig and stickleback, indicating an opposite tendency of recombination together or non-disjunction of these two genes during speciation. Further expression analysis showed an ubiquitous expression pattern of Ago members, oppositely a restricted expression pattern in gonads of the Piwi members, suggesting distinct potential roles of the porcine Argonaute genes.     

Expression and functional analysis of musashi-like genes in planarian CNS regeneration

The remarkable regenerative ability of planarians is made possible by a system of pluripotent stem cells. Recent molecular biological and ultrastructural studies have revealed that planarian stem cells consist of heterogeneous populations, which can be classified into several subsets according to their differential expression of RNA binding protein genes. In this study, we focused on planarian musashi family genes. Musashi encodes an evolutionarily conserved RNA binding protein known to be expressed in neural lineage cells, including neural stem cells, in many animals. Here, we investigated whether planarian musashi-like genes can be used as markers for detecting neural fate-restricted cells. Three musashi family genes, DjmlgA, DjmlgB and DjmlgC (Dugesia japonica musashi-like gene A, B, C), and Djdmlg (Dugesia japonica DAZAP-like/musashi-like gene) were obtained by searching a planarian EST database and 5′ RACE, and each was found to have two RNA recognition motifs. We analyzed the types of cells expressing DjmlgA, DjmlgB, DjmlgC and Djdmlg by in situ hybridization, RT-PCR and single-cell RT-PCR analysis. Although Djdmlg was expressed in X-ray-sensitive stem cells and various types of differentiated cells, expression of the other three musashi-like genes was restricted to neural cells, as we expected. Further detailed analyses yielded the unexpected finding that these three planarian musashi family genes were predominantly expressed in X-ray-resistant differentiated neurons, but not in X-ray-sensitive stem cells. RNAi experiments suggested that these planarian musashi family genes might be involved in neural cell differentiation after neural cell-fate commitment.     

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.     

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.     

The germ line and somatic stem cell gene Cniwi in the jellyfish Podocoryne carnea

In most animal phyla from insects to mammals, there is a clear division of somatic and germ line cells. This is however not the case in plants and some animal phyla including tunicates, flatworms and the basal phylum Cnidaria, where germ stem cells arise de novo from somatic cells. Piwi-like genes represent essential stem cell genes in diverse multicellular organisms. The cnidarian Piwihomolog Cniwiwas cloned from Podocoryne carnea, a hydrozoan with a full life cycle. CniwiRNA is present in all developmental stages with highest levels in the egg and the medusa. In the adult medusa, Cniwi expression is prominent in the gonads where it likely functions as a germ stem cell gene. The gene is also expressed, albeit at low levels, in differentiated somatic cells like the striated muscle of the medusa. Isolated striated muscle cells can be induced to transdifferentiate into smooth muscle cells which proliferate and differentiate into nerve cells. Cniwi expression is upregulated transiently after induction of transdifferentiation and again when the emerging smooth muscle cells proliferate and differentiate. The continuous low-level expression of an inducible stem cell gene in differentiated somatic cells may underlie the ability to form medusa buds from polyp cells and explain the extraordinary transdifferentation and regeneration potential of Podocoryne carnea.     

Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.     

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.     

Small RNAs in the animal gonad: Guarding genomes and guiding development

Germ cells must safeguard, apportion, package, and deliver their genomes with exquisite precision to ensure proper reproduction and embryonic development. Classical genetic approaches have identified many genes controlling animal germ cell development, but only recently have some of these genes been linked to the RNA interference (RNAi) pathway, a gene silencing mechanism centered on small regulatory RNAs. Germ cells contain microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs); these are bound by members of the Piwi/Argonaute protein family. piwi genes were known to specify germ cell development, but we now understand that mutations disrupting germline development can also affect small RNA accumulation. Small RNA studies in germ cells have revealed a surprising diversity of regulatory mechanisms and a unifying function for germline genes in controlling the spread of transposable elements. Future challenges will be to understand the production of germline small RNAs and to identify the full breadth of gene regulation by these RNAs. Progress in this area will likely impact biomedical goals of manipulating stem cells and preventing diseases caused by the transposition of mobile DNA elements.     

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.     

Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano

We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad formation and stem cell proliferation. Our results corroborate the exclusive nature of the flatworm stem cell system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells.