Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E 8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGC-resembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdm1 activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation.

In mammals, cell lineage specification is established at the blastocyst stage. At this stage, transcription factor Cdx2 represses pluripotency genes, thus promoting extraembryonic trophoblast fate. Recently, transcription factor Gata3 was shown to act in a parallel pathway in promoting trophoblast cell fate, suggesting that there are more factors working in the trophoblast lineage. Here, we report that the transcription factor Tcfap2c is expressed at a high level in the trophectoderm and is able to induce trophoblast fate in embryonic stem cells. Trophoblast fate induced by Tcfap2c does not require Cdx2 and vice versa, suggesting that the molecules act in alternative pathways. However, both Tcfap2c and Cdx2 are required for the upregulation of Elf5, a marker of trophoblast stem cell maintenance, suggesting that both factors are required for stable trophoblast induction. Tcfap2c-induced trophoblast-like cells are stable in long-term culture, indicating that they are capable of self-renewal. Tcfap2c-controlled trophoblast maintenance involves the induction of Cdx2 and the repression of the pluripotency factor Nanog. Tcfap2c-induced trophoblast-like cells differentiate to trophoblast derivatives in vitro and contribute to the trophectoderm in blastocysts in vivo. Taken together, these observations suggest that Tcfap2c and Cdx2 cooperate to override the pluripotency program and establish the extraembryonic trophoblast maintenance program in murine embryos.The earliest cell fate decision during mammalian development is the establishment of the first two cell lineages of the blastocyst prior to implantation. The inner cell mass (ICM) forms the embryo proper as well as extraembryonic endodermal components of the placenta, whereas the trophectoderm (TE) gives rise to the fetal portion of the placenta, a structure unique to mammalian development (11). Self-renewing embryonic stem cell (ESC) and trophoblast stem cell (TSC) lines have been derived from each of these lineages in vitro (10,23,36). TSCs exhibit the potential to differentiate into multiple trophoblastic cell types in vitro, participate in the normal development of chimeras, and contribute exclusively to the trophoblastic component of the placenta in vivo (28,36).At the genetic level, key factors that establish and maintain the TE lineage in the early embryo have been identified. Based on so far unknown positional information, the Hippo signaling pathway component YAP (Yes kinase-associated protein 1, a coactivator of Tead4) is phosphorylated by LATS (large tumor suppressor, a Ser/Thr kinase that belongs to the Ndr/LATS subfamily of protein kinase A/PKG/PKC kinases) and becomes cytoplasmic in the inner cells of the morula. In outer blastomeres, YAP remains in the nucleus and associates with and activates TEAD4 (26), which in turn transactivates the expression of the transcription factor CDX2 (caudal-related homeobox 2) (27, 40). CDX2 represses pluripotency markers such as Oct3/4 (Pou5f1) and Nanog (and vice versa), which leads to the maintenance of the restrict...

SummaryTrophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.

Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation.

In recent years, knowledge regarding the genetic and epigenetic programmes governing specification, maintenance and differentiation of the extraembryonic lineage has advanced substantially. Establishment and analysis of mice deficient in genes implicated in trophoblast lineage and the option to generate and manipulate murine stem cell lines from the inner cell mass and the trophectoderm in vitro represent major advances. The activating enhancer binding protein 2 (AP2) family of transcription factors is expressed during mammalian development and in certain malignant diseases. This article summarizes the data regarding expression and function of murine Tcfap2 and human TFAP2 in extraembryonic development and differentiation. It also presents a model integrating Tcfap2c into the framework of trophoblast development and highlights the requirement of Tcfap2c to maintain trophoblast stem cells. With regard to human trophoblast cell-lineage restriction, the role of TFAP2C in lineage specification and maintenance is speculated upon. Furthermore, an overview of target genes of AP2 in mouse and human affecting placenta development and function is provided and the evidence suggesting that defects in regulating TFAP2 members might contribute to placental defects is discussed.

In mammals, the first cell fate decision is initialized by cell polarization at the 8-to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals.Life starts with a single totipotent cell, followed by several cleavages and the specification of the earliest cell lineages at the blastocyst stage. The trophectoderm (TE) represents the extraembryonic trophoblast lineage and envelops the inner cell mass (ICM), which gives rise to the embryonic tissues and the primitive endoderm. Embryonic stem cells (ESCs) derived from the ICM are pluripotent because they differentiate into all embryonic tissues, including the germ line, but have lost the ability to form trophoblast derivatives (10). Likewise, trophoblast stem cells (TSCs) derived from the TE layer exclusively recapitulate the developmental potency of their parental lineage and contribute to all trophoblast cell types of the conceptus (40, 50).The first cell fate decision is initiated by cell polarization at the 8-to 16-cell stage, where inner and outer cells are differentiated from each other. Outer cells adopt the trophoblast fate, whereas the inner cell population gives rise to the ICM. Differentiation is established by the expression of lineage-specific transcription networks and fixed by epigenetic asymmetries at the late blastocyst stage (4,42,43,54).The caudal-related homeobox 2 factor, Cdx2, is regarded as one key transcription factor for TE specification and maintenance. Cdx2 is expressed in the outer blastomeres and later in TE (7, 32). Cdx2-null embryos lose trophectoderm integrity, and TSCs cannot be derived (48). The ectopic expression of Cdx2 in ESCs leads to lineage conversion to the extraembryonic trophoblast fate by the direct downregulation of pluripotency-associated transcription factors, such as Oct4 and Nanog, and the activation of the trophectodermal transcription network (5, 35). Oct4 (...

BackgroundThe transcription factor Tcfap2c has been demonstrated to be essential for various processes during mammalian development. It has been found to be upregulated in various undifferentiated tumors and is implicated with poor prognosis. Tcfap2c is reported to impinge on cellular proliferation, differentiation and apoptosis. However, the physiological consequences of Tcfap2c-expression remain largely unknown.Methodology/Principal FindingsTherefore we established a gain of function model to analyze the role of Tcfap2c in development and disease. Induction of the transgene led to robust expression in all tissues (except brain and testis) and lead to rapid mortality within 3–7 days. In the liver cellular proliferation and apoptosis was detected. Accumulation of microvesicular lipid droplets and breakdown of major hepatic metabolism pathways resulted in steatosis. Serum analysis showed a dramatic increase of enzymes indicative for hepatic failure. After induction of Tcfap2c we identified a set of 447 common genes, which are deregulated in both liver and primary hepatocyte culture. Further analysis showed a prominent repression of the cytochrome p450 system, PPARA, Lipin1 and Lipin2. These data indicate that in the liver Tcfap2c represses pathways, which are responsible for fatty acid metabolism. In the intestine, Tcfap2c expression resulted in expansion of Sox9 positive and proliferative active epithelial progenitor cells resulting in dysplastic growth of mucosal crypt cells and loss of differentiated mucosa.ConclusionsThe transgenic mice show that ectopic expression of Tcfap2c is not tolerated. Due to the phenotype observed, iTcfap2c-mice represent a model system to study liver failure. In intestine, Tcfap2c induced cellular hyperplasia and suppressed terminal differentiation indicating that Tcfap2c serves as a repressor of differentiation and inducer of proliferation. This might be achieved by the Tcfap2c mediated activation of Sox9 known to be expressed in intestinal and hepatic stem/progenitor cell populations.

Tfap2c is required for placental development and trophoblast stem cell maintenance. Deletion of Tfap2c results in early embryonic loss because of failure in placental development. We evaluated the effect of reduced Tfap2c expression on fetal outcome and placental development. Sixty percent of the heterozygous mice were lost directly after birth. Labyrinthine differentiation was impaired, as indicated by enhanced proliferation and inclusions of cobblestone-shaped cell clusters characterized by expression of Tfap2c and glycogen stores. Moreover, expression of marker genes such as Cdx2, Eomes, Gata3, and Ascl2 are decreased in the spongiotrophoblast and indicate a lowered stem cell potential. On Day 18.5 postcoitum, the labyrinth layer of Tfap2c(+/-) placentas exhibited massive hemorrhages in the maternal blood spaces; these hemorrhages might have contributed to the significantly reduced number of live-born pups. These morphological alterations were accompanied by a shift toward sinusoidal trophoblast giant cells as the cell subpopulation lining the maternal sinusoids and toward reduction in expression of the prolactin gene family member Prl2c2, a finding characteristic of the spiral arteries lining trophoblast cells. The trophoblast stem cells heterozygous for Tfap2c exhibited a reduction in the expression level of stem cell markers and in their proliferation and differentiation capacity but did not exhibit changes in marker genes of the trophoblast giant cell lineage. Taken together, these findings indicate that a reduction in the gene dosage of placental Tfap2c leads to morphological changes in the labyrinth at midgestation and in the maternal blood spaces during late pregnancy.