Anne Dupressoir

Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene

In most mammalian species, a key process of placenta development is the fusion of trophoblast cells into a highly specialized, multinucleated syncytiotrophoblast layer, through which most of the maternofetal exchanges take place. Little is known about this process, despite the recent identification of 2 pairs of envelope genes of retroviral origin, independently acquired by the human (syncytin-1 and syncytin-2) and mouse (syncytin-A and syncytin-B) genomes, specifically expressed in the placenta, and with in vitro cell–cell fusion activity. By generating knockout mice, we show here that homozygous syncytin-A null mouse embryos die in utero between 11.5 and 13.5 days of gestation. Refined cellular and subcellular analyses of the syncytin-A-deficient placentae disclose specific disruption of the architecture of the syncytiotrophoblast-containing labyrinth, with the trophoblast cells failing to fuse into an interhemal syncytial layer. Lack of syncytin-A-mediated trophoblast cell fusion is associated with cell overexpansion at the expense of fetal blood vessel spaces and with apoptosis, adding to the observed maternofetal interface structural defects to provoke decreased vascularization, inhibition of placental transport, and fetal growth retardation, ultimately resulting in death of the embryo. These results demonstrate that syncytin-A is essential for trophoblast cell differentiation and syncytiotrophoblast morphogenesis during placenta development, and they provide evidence that genes captured from ancestral retroviruses have been pivotal in the acquisition of new, important functions in mammalian evolution.

Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in placentation

The development of the emerging field of ‘paleovirology’ allows biologists to reconstruct the evolutionary history of fossil endogenous retroviral sequences integrated within the genome of living organisms and has led to the retrieval of conserved, ancient retroviral genes ‘exapted’ by ancestral hosts to fulfil essential physiological roles, syncytin genes being undoubtedly among the most remarkable examples of such a phenomenon. Indeed, syncytins are ‘new’ genes encoding proteins derived from the envelope protein of endogenous retroviral elements that have been captured and domesticated on multiple occasions and independently in diverse mammalian species, through a process of convergent evolution. Knockout of syncytin genes in mice provided evidence for their absolute requirement for placenta development and embryo survival, via formation by cell–cell fusion of syncytial cell layers at the fetal–maternal interface. These genes of exogenous origin, acquired ‘by chance’ and yet still ‘necessary’ to carry out a basic function in placental mammals, may have been pivotal in the emergence of mammalian ancestors with a placenta from egg-laying animals via the capture of a founding retroviral env gene, subsequently replaced in the diverse mammalian lineages by new env-derived syncytin genes, each providing its host with a positive selective advantage.

Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding

BackgroundThe yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core.ResultsHere, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner.ConclusionsWe have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals

BackgroundSyncytins are envelope genes of retroviral origin that have been co-opted by the host to mediate a specialized function in placentation. Two of these genes have already been identified in primates, as well as two distinct, non orthologous genes in rodents.ResultsHere we identified within the rabbit Oryctolagus cuniculus-which belongs to the lagomorpha order- an envelope (env) gene of retroviral origin with the characteristic features of a bona fide syncytin, that we named syncytin-Ory1. An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome first identified two candidate genes that were tested for their specific expression in the placenta by quantitative RT-PCR of RNA isolated from a large set of tissues. This resulted in the identification of an env gene with placenta-specific expression and belonging to a family of endogenous retroelements present at a limited copy number in the rabbit genome. Functional characterization of the identified placenta-expressed env gene after cloning in a CMV-driven expression vector and transient transfection experiments, demonstrated both fusogenic activity in an ex vivo cell-cell fusion assay and infectivity of pseudotypes. The receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin-1, i.e. the previously identified ASCT2 transporter. This was demonstrated by a co-culture fusion assay between hamster A23 cells transduced with an expression vector for ASCT2 and A23 cells transduced with syncytin-Ory1. Finally, in situ hybridization of rabbit placenta sections with a syncytin-Ory1 probe revealed specific expression at the level of the junctional zone between the placental lobe and the maternal decidua, where the invading syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The syncytin-Ory1 gene is found in Leporidae but not in Ochotonidae, and should therefore have entered the lagomorpha order 12-30 million years ago.ConclusionThe identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions retroviral infections have resulted in the independent capture of genes that have been positively selected for a convergent physiological role.

A pair of co-opted retroviral envelope syncytin genes is required for formation of the two-layered murine placental syncytiotrophoblast

In most mammalian species, a critical step of placenta development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast layer fulfilling essential fetomaternal exchange functions. Key insights into this process came from the discovery of envelope genes of retroviral origin, the syncytins, independently acquired by the human (syncytin-1 and -2), mouse (syncytin-A and -B), and rabbit (syncytin-Ory1) genomes, with fusogenic properties and placenta-specific expression. We previously showed that mouse syncytin-A is essential for the formation of one of the two syncytiotrophoblast layers and for embryo survival. Here, we have generated syncytin-B KO mice and demonstrate that syncytin-B null placenta displays impaired formation of syncytiotrophoblast layer II (ST-II), with evidence of unfused apposed cells, and enlargement of maternal lacunae disrupting the placenta architecture. Unexpectedly, syncytin-B null embryos are viable, with only limited late-onset growth retardation and reduced neonate number. Microarray analyses identified up-regulation of the connexin 30 gene in mutant placentae, with the protein localized at the fetomaternal interface, suggesting gap junction-mediated compensatory mechanisms. Finally, double-KO mice demonstrate premature death of syncytin-A null embryos if syncytin-B is deleted, indicating cooperation between ST-I and ST-II. These findings establish that both endogenous retrovirus-derived syncytin genes contribute independently to the formation of the two syncytiotrophoblast layers during placenta formation, demonstrating a major role of retroviral gene capture, through convergent evolution, to generate multiple placental structures. Although some are absolutely required for completion of pregnancy, others are still amenable to “epigenetic” compensations, thus illustrating the complexity of the molecular machinery that developed during placental evolution.

Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells

Mammalian genomes contain two main classes of retrotransposons, the well-characterized long and short interspersed nuclear elements, which account for ∼30% of the genome, and the long terminal repeat (LTR) retrotransposons, which resemble the proviral integrated form of retroviruses, except for the absence of an envelope gene in some cases. Genetic studies confirmed mobility of the latter class of elements in mice, with a high proportion of phenotypic mutations consequent to transposition of the intracisternal A particle (IAP) family of LTR retrotransposons. Using the mouse genome sequence and an efficient ex vivo retrotransposition assay, we identified functional, master IAP copies that encode all the enzymatic and structural proteins necessary for their autonomous transposition in heterologous cells. By introducing mutations, we found that the three genes gag, prt and pol are all required for retrotransposition and identified the IAP gene products by electron microscopy in the form of intracellular A-type particles in the transfected cells. These prototypic elements, devoid of an envelope gene, are the first LTR retrotransposons autonomous for transposition to be identified in mammals. Their high rates of retrotransposition indicate that they are potent insertional mutagens that could serve as safe (noninfectious) genetic tools in a large panel of cells.

Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora

Syncytins are envelope protein genes of retroviral origin that have been captured for a function in placentation. Two such genes have already been identified in simians, two distinct, unrelated genes have been identified in Muridae, and a fifth gene has been identified in the rabbit. Here, we searched for similar genes in the Laurasiatheria clade, which diverged from Euarchontoglires—primates, rodents, and lagomorphs—shortly after mammalian radiation (100 Mya). In silico search for envelope protein genes with full-coding capacity within the dog and cat genomes identified several candidate genes, with one common to both species that displayed placenta-specific expression, which was revealed by RT-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with precise proviral integration at a site common to dog and cat. Cloning of the gene for an ex vivo pseudotype assay showed fusogenicity on both dog and cat cells. In situ hybridization on placenta sections from both species showed specific expression at the level of the invasive fetal villi within the placental junctional zone, where trophoblast cells fuse into a syncytiotrophoblast layer to form the maternofetal interface. Finally, we show that the gene is conserved among a series of 26 Carnivora representatives, with evidence for purifying selection and conservation of fusogenic activity. The gene is not found in the Pholidota order and, therefore, it was captured before Carnivora radiation, between 60 and 85 Mya. This gene is the oldest syncytin gene identified to date, and it is the first in a new major clade of eutherian mammals.

Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation and likely contribute to the remarkable diversity of placental structures. Independent capture events have been identified in primates, rodents, lagomorphs, and carnivores, where they are involved in the formation of a syncytium layer at the fetomaternal interface via trophoblast cell–cell fusion. We searched for similar genes within the suborder Ruminantia where the placenta lacks an extended syncytium layer but displays a heterologous cell-fusion process unique among eutherian mammals. An in silico search for intact envelope genes within the Bos taurus genome identified 18 candidates belonging to five endogenous retrovirus families, with one gene displaying both placenta-specific expression, as assessed by quantitative RT-PCR analyses of a large panel of tissues, and conservation in the Ovis aries genome. Both the bovine and ovine orthologs displayed fusogenic activity by conferring infectivity on retroviral pseudotypes and triggering cell–cell fusion. In situ hybridization of placenta sections revealed specific expression in the trophoblast binucleate cells, consistent with a role in the formation—by heterologous cell fusion with uterine cells—of the trinucleate cells of the cow and the syncytial plaques of the ewe. Finally, we show that this gene, which we named “Syncytin-Rum1,” is conserved among 16 representatives of higher ruminants, with evidence for purifying selection and conservation of its fusogenic properties, over 30 millions years of evolution. These data argue for syncytins being a major driving force in the emergence and diversity of the placenta.

Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials

Significance Syncytins are “captured” genes of retroviral origin, corresponding to the fusogenic envelope gene of endogenized retroviruses. They are present in a series of eutherian mammals, including humans and mice where they play an essential role in placentation. Here we show that marsupials—which diverged from eutherian mammals ∼190 Mya but still possess a primitive, short-lived placenta (rapidly left by the embryo for development in an external pouch)—have also captured such genes. The present characterization of the syncytin-Opo1 gene in the opossum placenta, together with the identification of two additional endogenous retroviral envelope gene captures, allow a recapitulation of the natural history of these unusual genes and definitely extends their “symbiotic niche” to all clades of placental mammals. Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials—which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya—also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell–cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto–maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses—displaying strong expression in the uterine glands where retroviral particles can be detected—plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades.

Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs

Significance Syncytins are genes of retroviral origin that have been captured by their host as symbionts for a function in placentation. They can mediate cell–cell fusion, consistent with their ancestral retroviral envelope gene status, and are involved in fusion of mononucleate trophoblast cells to form the syncytial layer—the syncytiotrophoblast—of the feto–maternal interface. We proposed that such genes have been pivotal for the emergence of placental mammals from egg-laying animals and should be present all along the Placentalia radiation. We searched for syncytins in a superorder of eutherian mammals that emerged ancestrally during the Cretaceous terrestrial revolution and identified syncytin-Ten1, conserved over millions years of evolution of the Afrotherian tenrecs, regarded as among the most primitive of living mammals. Syncytins are fusogenic envelope (env) genes of retroviral origin that have been captured for a function in placentation. Syncytins have been identified in Euarchontoglires (primates, rodents, Leporidae) and Laurasiatheria (Carnivora, ruminants) placental mammals. Here, we searched for similar genes in species that retained characteristic features of primitive mammals, namely the Malagasy and mainland African Tenrecidae. They belong to the superorder Afrotheria, an early lineage that diverged from Euarchotonglires and Laurasiatheria 100 Mya, during the Cretaceous terrestrial revolution. An in silico search for env genes with full coding capacity within a Tenrecidae genome identified several candidates, with one displaying placenta-specific expression as revealed by RT-PCR analysis of a large panel of Setifer setosus tissues. Cloning of this endogenous retroviral env gene demonstrated fusogenicity in an ex vivo cell–cell fusion assay on a panel of mammalian cells. Refined analysis of placental architecture and ultrastructure combined with in situ hybridization demonstrated specific expression of the gene in multinucleate cellular masses and layers at the materno–fetal interface, consistent with a role in syncytium formation. This gene, which we named “syncytin-Ten1,” is conserved among Tenrecidae, with evidence of purifying selection and conservation of fusogenic activity. To our knowledge, it is the first syncytin identified to date within the ancestrally diverged Afrotheria superorder.