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Transcriptomic signatures of villous cytotrophoblast and syncytiotrophoblast in term human placenta.

ABSTRACT: During pregnancy, the placenta ensures multiple functions, which are directly involved in the initiation, fetal growth and outcome of gestation. The placental tissue involved in maternal-fetal exchanges and in synthesis of pregnancy hormones is the mononucleated villous cytotrophoblast (VCT) which aggregates and fuses to form and renew the syncytiotrophoblast (ST). Knowledge of the gene expression pattern specific to this endocrine and exchanges tissue of human placenta is of major importance to understand functions of this heterogeneous and complex tissue. Therefore, we undertook a global analysis of the gene expression profiles of primary cultured-VCT (n=6) and in vitro-differentiated-ST (n=5) in comparison with whole term placental tissue from which mononucleated VCT were isolated. A total of 880 differentially expressed genes (DEG) were observed between VCT/ST compared to whole placenta, and a total of 37 and 137 genes were significantly up and down-regulated, respectively, in VCT compared to ST. The 37 VCT-genes were involved in cellular processes (assembly, organization, and maintenance), whereas the 137 ST-genes were associated with lipid metabolism and cell morphology. In silico, all networks were linked to 3 transcriptional regulators (PPARγ, RARα and NR2F1) which are known to be essential for trophoblast differentiation. Furthermore, a subset of DEG were validated by RT-qPCR or by immunohistochemistry. To conclude, recognition of these pathways is fundamental to increase our understanding of the molecular basis of human trophoblast differentiation. The present study provides for the first time a gene expression signature of the VCT and ST compared to their originated term human placental tissue.

ORGANISM(S): Homo sapiens

PROVIDER: GSE79333 | GEO | 2016/12/31

SECONDARY ACCESSION(S): PRJNA315528

REPOSITORIES: GEO

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Project description:In most mammalian species, a critical step of placental development is the fusion of trophoblast cell into a multinucleated syncytiotrophoblast layer, which separates the fetal and maternal blood circulations. Advancement in understanding this process came from the identification of two pairs of envelope genes of retroviral origin, independently acquired by the human (syncytin-1 and M-^V2) and mouse (syncytin-A and M-^VB) genomes, specifically expressed in the placenta and with in vitro cell-cell fusion activity. We previously showed that syncytin-A is involved in the formation of one of the two syncytiotrophoblast layer of the mouse placenta -ST-I , facing the maternal lacuna- with syncytin-A null embryos dying at mid-gestation, and their placenta disclosing impaired formation of the ST-I layer. Here by generating syncytin-B KO mice, we demonstrated that syncytin-B null placenta have defects in formation of the syncytiotrophoblast layer-II facing the fetal blood vessels, with refined electron microscopy analyses showing evidence of unfused apposed cells. Lack of trophoblast fusion is associated with signs of trophoblast degeneration and with formation of huge maternal blood lacuna, ultimately disrupting the architecture of the labyrinth layer. These alterations did not result in complete abortion of syncytin-B null embryos, at variance with the syncytin-A KO phenotype, but in a reduced proportion of homozygous syncytin-B knockout individuals at birth, with evidence of late-onset embryonic growth retardation. Double knockout mice experiments demonstrate a premature death of syncytin-A null embryos if syncytin-B is deleted, thus strongly suggesting cooperative involvement of both syncytiotrophoblast layer-I and layer-II for the structural and functional integrity of the maternofetal interface and exchanges. Finally, a microarray analysis of wild-type and syncytin-B null placenta transcripts disclosed alterations in gene expression level for only a very limited number of genes, with the largest change (a 7-fold induction in the syncytin-B null placenta) observed for the connexin-30 gene. Immunohistochemistry further localized the connexin-30 protein in the labyrinth zone of mutant placenta, at the level of the fetomaternal interface. It is proposed that this might correspond to a compensatory process whereby disruption of syncytialization in syncytin-B knockout placenta is counteracted by an enhanced, gap junction mediated, cell-cell communication. These data demonstrate that syncytin-B is required for ST-II syncytial differentiation and the functional integrity of the labyrinth. Altogether, these findings definitively demonstrate that the two endogenous retroviral syncytin-A and -B Env genes contribute independently to the formation of the two syncytiotrophoblast layers during placenta formation, and provide additional insights into the subtle mechanisms of syncytiotrophoblast differentiation in the mouse. Experimental design: Placenta RNA of embryos homozygous for the syncytin-B deletion (SynB-/-) were compared to placental RNA of wild-type embryos (SynB+/+) of the same litter. Two stages of gestation, day 12 and d14, were analyzed. At day 12, 3 litters were obtained; for two of them, the RNAs were analyzed individually, and for the third one, placenta RNAs of the wild-type genotype were pooled. At day 14, one litter was obtained; RNA samples with the same genotype were pooled

Project description:Healthy placental development is essential for reproductive success; failure of the feto-maternal interface results in preeclampsia and intrauterine growth retardation. We found that grainyhead-like 2 (GRHL2), a CP2-type transcription factor, is highly expressed in chorionic trophoblast cells, including basal chorionic trophoblast (BCT) cells located at the chorioallantoic interface in murine placentas. Placentas from Grhl2-deficient mouse embryos displayed defects in BCT cell polarity and basement membrane integrity at the chorioallantoic interface, as well as a severe disruption of labyrinth branchingmorphogenesis.Selective Grhl2 inactivation only in epiblastderived cells rescued all placental defects but phenocopied intraembryonic defects observed in global Grhl2 deficiency, implying the importance of Grhl2 activity in trophectoderm-derived cells. ChIPseq identified 5282 GRHL2 binding sites in placental tissue. By integrating these data with placental gene expression profiles, we identified direct and indirect Grhl2 targets and found a marked enrichment of GRHL2 binding adjacent to genes downregulated in Grhl2−/− placentas, which encoded known regulators of placental development and epithelial morphogenesis. These genes included that encoding the serine protease inhibitor Kunitz type 1 (Spint1), which regulates BCT cell integrity and labyrinth formation. In human placenta, we found that human orthologs of murine GRHL2 and its targets displayed co-regulation and were expressed in trophoblast cells in a similar domain as in mouse placenta. Our data indicate that a conserved Grhl2-coordinated gene network controls trophoblast branching morphogenesis, thereby facilitating development of the site of feto-maternal exchange. This might have implications for syndromes related to placental dysfunction. In vivo genome-wide examination of binding sites of the transcription factor GRHL2 by ChIP-seq using wild-type murine E17.5 placenta tissue. Two samples in total: one GRHL2 ChIP sample and one IgG ChIP sample using wild-type placentas tissue as antibody control.

Project description:Purpose: Syncytiotrophoblast (STB) is a multi-nucleated, terminally differentiated syncytium that covers the surface of the villous placenta and forms the major interface with maternal blood. It releases placental hormones and plays a primary role in exchange of gases, nutrients and waste products. Alterations in STB development and turnover have been implicated in placental diseases, including preeclampsia (PE). In vitro cell models are badly needed to study STB development and physiology due to inaccessibility to placental tissues during gestation. To establish in vitro STB model system, we generate STB and its mononucleated precursors from human embryonic stem cells (hESC) and profile for RNA content by RNAseq. Methods: H1 Human ESC (WA01) were treated with BMP4, the ALK4/5/7 inhibitor (A83-01), and the FGF2 signaling inhibitor (PD173074) (BAP) to direct them to the trophoblast lineage and provided both STB and extravillous trophoblast. Syncytial areas emerged at day 8 BAP treatment ranged in diameter from ~40 Âµm to > 100 Âµm. The intact syncytial areas were isolated by sieving successively through 70 Î¼m and 40 Î¼m mesh cell strainers. The captured cells are recovered by inverting the strainer and rinsing with culture medium to separate large (>70 Î¼m) and middle size cell sheets (40-70 Î¼m). The fraction that passes through both sieves represents cells of smallest diameter (< 40 Î¼m), presumably cytotrophoblast. Total 12 RNA samples from triplicate three size-fractioned BAP treated and three untreated hESC cultured in a FGF2 supplemented medium in parallel were analyzed. Results: The larger > 70 Î¼m areas stained positively for STB markers while ultrastructural analysis clearly revealed multi-nuclear cells with an extensive cytoplasm containing many prominent secretion granules. The larger STB areas also had a larger DNA content that > 70 Î¼m fraction contained 37 times more nuclear content and 40-70 Âµm fraction did 16 times more. Compared to the < 40 Î¼m cell fraction, these larger cells over-expressed a full repertoire of genes characteristic of STB, e.g. CGA, CGB, PGF, ERVW1, GCM1. The smallest cell fraction had a DNA content consistent with mononuclear diploid cells, contained few secretory granules, and were only weakly positive for STB markers. Conclusion: The data are consistent with the > 70 Î¼m cells being mature STB, while the intermediate fraction may represent a precursor population. Human ESC directed along the trophoblast lineage by BAP treatment offers a useful model for following STB formation in vitro and suggest that this protocol may have utility in studying the basis of certain placental diseases, especially preeclampsia, where placental tissue isolated at term or after pregnancy terminations can only offer limited information. Three size fraction mRNA profiles of syncytial areas emerged at day 8 BAP treatment of hESC were generated by deep sequencing along with untreated hESC, in triplicate, using Illumina HiSeq 2500.

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Transcriptomic signatures of villous cytotrophoblast and syncytiotrophoblast in term human placenta.

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