An endogenous retroviral envelope syncytin and its PNAS PLUS cognate receptor identified in the viviparous placental Mabuya lizard Guillaume Cornelisa,b,1,2, Mathis Funka,b,1, Cécile Vernocheta,b, Francisca Lealc,3, Oscar Alejandro Tarazonac,4, Guillaume Meuriced, Odile Heidmanna,b, Anne Dupressoira,b, Aurélien Mirallese, Martha Patricia Ramirez-Pinillac, and Thierry Heidmanna,b,5 aUnité Physiologie et Pathologie Moléculaires des Rétrovirus Endogènes et Infectieux, CNRS UMR 9196, Gustave Roussy, Villejuif, F-94805, France; bUMR 9196, Université Paris-Sud, Orsay, F-91405, France; cLaboratorio de Biologia Reproductiva de Vertebrados, Escuela de Biologia, Universidad Industrial de Santander, 680002 Bucaramanga, Colombia; dPlateforme de Bioinformatique, INSERM US23/CNRS UMS3655, Gustave Roussy, Villejuif, F-94805, France; and eInstitut de Systématique, Evolution, Biodiversité, Muséum National d’Histoire Naturelle, CNRS UPMC EPHE, Sorbonne Universités, Paris, F-75005, France Edited by R. Michael Roberts, University of Missouri-Columbia, Columbia, MO, and approved October 26, 2017 (received for review August 23, 2017) Syncytins are envelope genes from endogenous retroviruses that Remarkably, placental structures are not restricted to mamma- have been captured during evolution for a function in placentation. lian species. Placentation emerged independently and in a sto- They have been found in all placental mammals in which they have chastic manner in several groups of vertebrates, with the noticeable been searched, including marsupials. Placental structures are not exception of birds (reviewed in refs. 16 and 17). In particular, restricted to mammals but also emerged in some other vertebrates, complex placentas have been described in some South American most frequently in lizards, such as the viviparous Mabuya Scincidae. or African species of Scincidae lizards (18–21; reviewed in refs. 16, Here, we performed high-throughput RNA sequencing of a Mabuya 22, and 23). In one Scincidae genus, Mabuya, placental structures placenta transcriptome and screened for the presence of retroviral env form specialized regions very similar to those found in mammals, genes with a full-length ORF. We identified one such gene, which we e.g., the placentome where maternal and invasive fetal tissues are named “syncytin-Mab1,” that has all the characteristics expected for highly folded and interdigitated. Ultrastructural analysis of this a syncytin gene. It encodes a membrane-bound envelope protein with region further revealed the presence of a syncytial structure at the MICROBIOLOGY fusogenic activity ex vivo, is expressed at the placental level as materno–fetal interface, as observed in numerous mammalian revealed by in situ hybridization and immunohistochemistry, and is species. However, in contrast to most mammals, the syncytial layer conserved in all Mabuya species tested, spanning over 25 My of evo- of the Mabuya placenta is formed by maternal epithelial cells lution. Its cognate receptor, required for its fusogenic activity, was rather than by fetal trophoblast cells, which remain individualized searched for by a screening assay using the GeneBridge4 human/Chi- nese hamster radiation hybrid panel and found to be the MPZL1 gene, Significance previously identified in mammals as a signal-transducing transmem- brane protein involved in cell migration. Together, these results show Retroviral envelope gene capture and exaptation for a placen- that syncytin capture is not restricted to placental mammals, but can tal function has been demonstrated in mammals. Remarkably, also take place in the rare nonmammalian vertebrates in which a vi- placental structures have also emerged on rare occasions in viparous placentotrophic mode of reproduction emerged. It suggests nonmammalian vertebrates, resulting in related modes of re- that similar molecular tools have been used for the convergent evo- production. The Mabuya lizard, which emerged 25 Mya, pos- lution of placentation in independently evolved and highly distant sesses a placenta closely related to that of mammals. Here, vertebrates. we identified a specific retroviral envelope gene capture that shows all the characteristic features of a bona fide mammalian endogenous retrovirus | envelope protein | placenta | syncytin | receptor syncytin, being conserved in Mabuya evolution, expressed in the placenta, and fusogenic. Together with the present identi- yncytins are “captured” genes of retroviral origin that corre- fication of its cognate receptor, these results show that syncytin Sspond to the envelope gene of ancestrally endogenized retro- capture is not restricted to mammals and is likely to be a major viruses. These genes encode fusogenic proteins that are involved in driving force for placenta emergence. the formation, by cell–cell fusion, of the syncytiotrophoblast at the placental materno–fetal interface in eutherian mammals (reviewed Author contributions: G.C., M.F., and T.H. designed research; G.C., M.F., and C.V. per- in refs. 1 and 2). Furthermore, genetically modified mice in which formed research; F.L., O.A.T., A.M., and M.P.R.-P. collected and processed live biological materials and samples; G.M. contributed analytic tools; G.C., M.F., C.V., G.M., O.H., A.D., the two syncytin genes, syncytin-A and syncytin-B, were knocked out A.M., M.P.R.-P., and T.H. analyzed data; and G.C., M.F., and T.H. wrote the paper. showed deficiencies in placental development, with altered struc- The authors declare no conflict of interest. ture of the materno–fetal interface resulting in inhibition of growth This article is a PNAS Direct Submission. or death of the embryo at midgestation (3, 4). Syncytins have been found in all placental mammals in which they have been searched, Published under the PNAS license. with independently captured syncytins occurring across all major Data deposition: RNA sequencing data are deposited in the European Nucleotide Archive, www.ebi.ac.uk/ena (accession no. ERA1116158). Mabuya sequences described in this pa- clades of placental mammals, including Euarchontoglires (primates, per (Mabuya MPZL1, syncytin-Mab1, and Mab-Env2–Mab-Env4) have been deposited in rodents, and lagomorphs), Laurasiatherians (ruminants and carni- the GenBank database (accession nos. MG254887–MG254891, respectively). vores), Afrotherians (tenrec), and even Marsupials (opossum) (Fig. 1G.C. and M.F. contributed equally to this work. 1) (5–15). This has led to the proposal that these genes, which are 2Present address: Department of Genetics, Stanford University, Stanford, CA 94305. absolutely required for placentation as shown by the knockout mice 3Present address: Department of Biology, University of Florida, Gainesville, FL 32611. experiments, are most likely involved in the emergence and evo- 4Present address: Department of Molecular Genetics and Microbiology, University of lution of placental mammals from egg-laying animals (1). Analysis Florida, Gainesville, FL 32611. of the conservation of these genes further indicates that they have 5To whom correspondence should be addressed. Email: [email protected]. been subjected to purifying selection in the course of evolution, as This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. expected for any bona fide cellular gene. 1073/pnas.1714590114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1714590114 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 Tenrecidae Syn-Ten1 implicated in cell-migration processes (29–32). This identification Proboscidae of a captured syncytin and its receptor in a distant viviparous lizard Dasypodidae with a placentotrophic mode of reproduction shows that syncytin Bradypodidae capture is not restricted to mammals and is likely to be a major Lagomorpha Syn-Ory1 driving force for placenta emergence and evolution. Rodentia Syn-A,-B,-Mar1 eutherian Haplorrhini Syn-1, -2 mammals Results MAMMALIAN Strepsirrhini High-Throughput Sequencing and in Silico Search for Retroviral Env PLACENTA Ruminantia Syn-Rum1 Genes Within the Mabuya Placental Transcriptome. Placental RNA Perissodactyla was extracted from pregnant Andean Mabuya sp. IV females Carnivora Syn-Car1 captured in the wild (Methods). The transcriptome of a placenta at Insectivora embryonic development stage 35 (as defined in ref. 33), presenting Didelphimorphia Syn-Opo1 marsupials a well-established syncytium, was determined by the French Na- Diprotodontia tional Sequencing Center (Genoscope, Evry, France). Placental Monotremata transcripts were assembled using de novo assembly methods in absence of a reference genome (Methods). Transcriptome recon- Gallus gallus (chicken) birds struction identified 72,763 transcripts ranging from 123 bp to Mabuya lizards 24.4 kb [N50 (the length N for which 50% of all bases in the Anolis carolinensis sequences are in a sequence of length L < N) = 2,869 bp; median Danio rerio (zebrafish) size = 1.1 kb], 26,690 of which (36.7%) could be positively Latimeria chalumnae bony fish (coelacanth) matched to a known protein gene from the Refseq Vertebrate 50100150200250300350400 0 Mya database. A set of 21,253 transcripts shows significant expression (more than two tags per million) (34), 16,398 of which were Devonian CarboniferousPermianTriassic Jurassic Cretaceous Tertiary positively annotated (77.2%) and correspond to 13,189 unique genes, among which 10,079 were nonhousekeeping genes. This subset of transcripts contains a range of genes annotated as “placental” genes with key biological functions during