Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta

The placenta is composed of multiple trophoblast cell types that have diverse endocrine, vascular and nutrient transport functions. We have developed a transgenic system to investigate the developmental and functional roles of specific cell types using conditional expression of a cytotoxin to induce cell ablation in transgenic mice. The Tpbpa gene is expressed in ectoplacental cone cells starting between embryonic days (E) 7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. Tpbpa-positive cells are progenitors of many trophoblast subtypes including three subtypes of trophoblast giant cells (TGCs) and glycogen trophoblast cells. We used a Cre recombinase transgene driven by the Tpbpa promoter to irreversibly activate a diphtheria toxin A (DTA) transgene. Cre/DTA double transgenic placentas showed dramatic reduction of Tpbpa-positive spongiotrophoblast cells by E10.5 and conceptuses died by ~ E11.5. The number of cells associated with maternal blood spaces, spiral artery TGCs (SpA-TGCs) and canal TGCs, and glycogen trophoblast cells were reduced. The loss of these specific trophoblast subtypes, especially SpA-TGCs, was correlated with a decrease in maternal spiral artery diameters, indicating a critical role of these cells in modulating the maternal vasculature. In contrast, parietal TGCs were not significantly reduced by progenitor cell ablation, suggesting that there is compensatory growth of this population and indeed a population of Ascl2 (Mash2)-positive/Tpbpa-negative cells was increased in the spongiotrophoblast layer in the Cre/DTA double transgenics. Our work demonstrates that the Tpbpa-positive lineage is essential for placental function and particularly critical for maternal vasculature remodeling.     

Determinants of trophoblast lineage and cell subtype specification in the mouse placenta

Cells of the trophoblast lineage make up the epithelial compartment of the placenta, and their rapid development is essential for the establishment and maintenance of pregnancy. A diverse array of specialized trophoblast subtypes form throughout gestation and are responsible for mediating implantation, as well as promotion of blood to the implantation site, changes in maternal physiology, and nutrient and gas exchange between the fetal and maternal blood supplies. Within the last decade, targeted mutations in mice and the study of trophoblast stem cells in vitro have contributed greatly to our understanding of trophoblast lineage development. Here, we review recent insights into the molecular pathways regulating trophoblast lineage segregation, stem cell maintenance, and subtype differentiation.     

Development of the hemochorial maternal vascular spaces in the placenta through endothelial and vasculogenic mimicry

The maternal vasculature within the placenta in primates and rodents is unique because it is lined by fetal cells of the trophoblast lineage and not by maternal endothelial cells. In addition to trophoblast cells that invade the uterine spiral arteries that bring blood into the placenta, other trophoblast subtypes sit at different levels of the vascular space. In mice, at least five distinct subtypes of trophoblast cells have been identified which engage maternal endothelial cells on the arterial and venous frontiers of the placenta, but which also form the channel-like spaces within it through a process analogous to formation of blood vessels (vasculogenic mimicry). These cells are all large, post-mitotic trophoblast giant cells. In addition to assuming endothelial cell-like characteristics (endothelial mimicry), they produce dozens of different hormones that are thought to regulate local and systemic maternal adaptations to pregnancy. Recent work has identified distinct molecular pathways in mice that regulate the morphogenesis of trophoblast cells on the arterial and venous sides of the vascular circuit that may be analogous to specification of arterial and venous endothelial cells.     

Activin promotes differentiation of cultured mouse trophoblast stem cells towards a labyrinth cell fate

Prolonged maintenance of trophoblast stem (TS) cells requires fibroblast growth factor (FGF) 4 and embryonic fibroblast feeder cells or feeder cell-conditioned medium. Previous studies have shown that TGF-β and Activin are sufficient to replace embryonic fibroblast-conditioned medium. Nodal, a member of the TGF-β superfamily, is also known to be important in vivo for the maintenance of TS cells in the developing placenta. Our current studies indicate that TS cells do not express the Nodal co-receptor, Cripto, and do not respond directly to active Nodal in culture. Conversely, Activin subunits and their receptors are expressed in the placenta and TS cell cultures, with Activin predominantly expressed by trophoblast giant cells (TGCs). Differentiation of TS cells in the presence of TGC-conditioned medium or exogenous Activin results in a reduction in the expression of TGC markers. In line with TGC-produced Activin representing the active component in TGC-conditioned medium, this differentiation-inhibiting effect can be reversed by the addition of follistatin. Additional experiments in which TS cells were differentiated in the presence or absence of exogenous Activin or TGF-β show that Activin but not TGF-β results in the maintenance of expression of TS cell markers, prolongs the expression of syncytiotrophoblast markers, and significantly delays the expression of spongiotrophoblast and TGC markers. These results suggest that Activin rather than TGF-β (or Nodal) acts directly on TS cells influencing both TS cell maintenance and cell fate, depending on whether the cells are also exposed to FGF4.     

Genetic insights into trophoblast differentiation and placental morphogenesis

The placenta is comprised of an inner vascular network covered by an outer epithelium, called trophoblast, all designed to promote the delivery of nutrients to the fetus. Several specialized trophoblast cell subtypes arise during development to promote this function, including cells that invade the uterus to promote maternal blood flow to the implantation site, and other cells that fuse into a syncytium, expand and fold to increase the surface area for efficient transport. Mutation of many genes in mice results in embryonic mortality or fetal growth restriction due to defects in placental development. Several important principles about placental development have emerged from these studies. First, distinct molecular pathways regulate the differentiation of the various trophoblast cell subtypes. Second, trophoblast proliferation, differentiation and morphogenesis are highly regulated by interactions with adjacent cell types. Finally, the specific classes of mutant phenotypes observed in the placenta of knockout mice resemble those seen in humans that are associated with preeclampsia and intrauterine growth restriction.     

Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta

Mammalian embryos have an intimate relationship with their mothers, particularly with the placental vasculature from which embryos obtain nutrients essential for growth. It is an interesting vascular bed because maternal vessel number and diameter change dramatically during gestation and, in rodents and primates, the terminal blood space becomes lined by placental trophoblast cells rather than endothelial cells. Molecular genetic studies in mice aimed at identifying potential regulators of these processes have been hampered by lack of understanding of the anatomy of the vascular spaces in the placenta and the general nature of maternal-fetal vascular interactions. To address this problem, we examined the anatomy of the mouse placenta by preparing plastic vascular casts and serial histological sections of implantation sites from embryonic day (E) 10.5 to term. We found that each radial artery carrying maternal blood into the uterus branched into 5-10 dilated spiral arteries located within the metrial triangle, populated by uterine natural killer (uNK) cells, and the decidua basalis. The endothelial-lined spiral arteries converged together at the trophoblast giant cell layer and emptied into a few straight, trophoblast-lined "canals" that carried maternal blood to the base of the placenta. Maternal blood then percolated back through the intervillous space of the labyrinth toward the maternal side of the placenta in a direction that is countercurrent to the direction of the fetal capillary blood flow. Trophoblast cells were found invading the uterus in two patterns. Large cells that expressed the trophoblast giant cell-specific gene Plf (encoding Proliferin) invaded during the early postimplantation period in a pattern tightly associated with spiral arteries. These peri/endovascular trophoblast were detected only approximately 150-300 microm upstream of the main giant cell layer. A second type of widespread interstitial invasion in the decidua basalis by glycogen trophoblast cells was detected after E12.5. These cells did not express Plf, but rather expressed the spongiotrophoblast-specific gene Tpbp. Dilation of the spiral arteries was obvious between E10.5 and E14.5 and was associated with a lack of elastic lamina and smooth muscle cells. These features were apparent even in the metrial triangle, a site far away from the invading trophoblast cells. By contrast, the transition from endothelium-lined artery to trophoblast-lined (hemochorial) blood space was associated with trophoblast giant cells. Moreover, the shaping of the maternal blood spaces within the labyrinth was dependent on chorioallantoic morphogenesis and therefore disrupted in Gcm1 mutants. These studies provide important insights into how the fetoplacental unit interacts with the maternal intrauterine vascular system during pregnancy in mice.     

Placental lactogen production and functional differentiation of rat trophoblast cells in vitro

Cells from the labyrinth region of the developing rat chorioallantoic placenta were able to differentiate in vitro into cells capable of producing placental lactogen. Progesterone selectively inhibited placental lactogen production by labyrinth cell cultures undergoing differentiation but had no apparent effect on lactogen production by mature trophoblast giant cells. The measurement of placental lactogen production is a useful method for monitoring the functional differentiation of rat trophoblast cells in vitro.     

Maternal hypoxia activates endovascular trophoblast cell invasion

Oxygen is a critical regulator of placentation. Early placental development occurs in a predominantly low oxygen environment and is, at least partially, under the control of hypoxia signaling pathways. In the present study, in vivo hypobaric hypoxia was used as an experimental tool to delineate hypoxia-sensitive events during placentation. Pregnant rats were exposed to the equivalent of 11% oxygen between days 6.5 and 13.5 of gestation. Pair-fed pregnant animals exposed to ambient conditions were included as a control group. Uterine mesometrial blood vessels in the hypoxia-exposed animals were greatly expanded and some contained large cuboidal cells that were positive for cytokeratin and other markers characteristic of invasive trophoblast cells. Unlike later in gestation, the route of trophoblast cell invasion in the hypoxia-exposed animals was restricted to endovascular, with no interstitial invasion observed. Hypoxia-activated endovascular trophoblast invasion required exposure to hypoxia from gestation day 8.5 to day 9.5. Activation of the invasive trophoblast lineage was also associated with an enlargement of the junctional zone of the chorioallantoic placenta, a source of invasive trophoblast cell progenitors. In summary, maternal hypoxia during early stages of placentation activates the invasive endovascular trophoblast cell lineage and promotes uterine vascular remodeling.     

Molecular mechanisms of trophoblast survival: from implantation to birth

Fetal development depends upon a coordinated series of events in both the embryo and in the supporting placenta. The initial event in placentation is appropriate lineage allocation of stem cells followed by the formation of a spheroidal trophoblastic shell surrounding the embryo, facilitating implantation into the uterine stroma and exclusion of oxygenated maternal blood. In mammals, cellular proliferation, differentiation, and death accompany early placental development. Programmed cell death is a critical driving force behind organ sculpturing and eliminating abnormal, misplaced, nonfunctional, or harmful cells in the embryo proper, although very little is known about its physiological function during placental development. This review summarizes current knowledge of the cell death patterns and molecular pathways governing the survival of cells within the blastocyst, with a focus on the trophoblast lineage prior to and after implantation. Particular emphasis is given to human placental development in the context of normal and pathological conditions. As molecular pathways in humans are poorly elucidated, we have also included an overview of pertinent genetic animal models displaying defects in trophoblast survival.     

PTHrP induces changes in cell cytoskeleton and E-cadherin and regulates Eph/Ephrin kinases and RhoGTPases in murine secondary trophoblast cells

The differentiation of murine trophoblast giant cells (TGCs) is well characterised at the molecular level and, to some extent, the cellular level. Currently, there is a rudimentary understanding about factors regulating the cellular differentiation of secondary TGCs. Using day 8.5 p.c.-ectoplacental cone (EPC) explant in serum-free culture, we have found parathyroid hormone-related protein (PTHrP) to regulate cellular changes during TGC differentiation. PTHrP greatly stimulated the formation and organisation of actin stress fibres and actin expression in trophoblast outgrowth. This coincided with changing cell shape into a flattened/fibroblastic morphology, suppression of E-cadherin expression, and increased cell spreading in culture. PTHrP also increased the nuclear staining of beta-catenin and, similar to activator protein-2gamma (AP-2gamma), showed microtubule-dependent nuclear localisation in vitro. These cellular and behavioural changes correlated with changes in the expression of RhoGTPases and in both expression and phosphorylation of Eph/Ephrin kinases. The effects of PTHrP on trophoblast cellular differentiation were abolished after blocking its action. In conclusion, PTHrP provides an excellent example of the extrinsic factors that, through their network of activities, plays an important role in cellular differentiation of secondary TGCs.     

The existence during gestation of an immunological buffer zone at the interface between maternal and foetal tissues.

In mammalian pregnancy the trophoblast normally constitutes an uninterrupted boundary of foetal tissue in immediate contact with maternal tissue, including blood in some species, and is the decisive immunological barrier to rejection of the foetus as an allograft. The ability of the trophoblast to function as a barrier evidently results from its capacity to resist immunological attack by either alloantibody or alloimmune cells and to prevent immunocompetent cells from reaching and damaging the foetus but, as yet, there is no general agreement regarding the means by which it exercises these functions. In view of the dramatic hormonal changes that occur during pregnancy and the undisputed involvement of trophoblast in these endocrine events, the possibility exists of an interaction between the hormones of pregnancy and the immunological phenomena. The present account furnishes evidence that endocrine activity at the maternal surface of the trophoblast, the presumptive site of the immunological frontier between foetus and mother, may be a factor in its local survival at implantation. The placental hormones so far known that are capable of blocking the antigen receptor sites of the mother's lymphocytes and thus preventing the latter from reacting with the foetal antigens are the glycoprotein, human chorionic gonadotrophin (HCG) and the polypeptide hormone, human chorionic somatomammotrophin (HCS) or human placental lactogen (HPL), both of which are specific to the human placenta. The origin of these hormones, their spatial distribution and their probable interaction with placental steroid hormones are discussed. It is argued that the place of highest concentration of these hormones is on the surface of the syncytial microvilli and the adjacent caviolae of the apical plasma membrane, as well as on the surfaces of the persisting cytotrophoblastic cells of the basal plate (cytotrophoblastic shell), the cell islands and the septa-precisely where the immunological challenge of the foetal allograft to the maternal host occurs. An explanation is offered for the continuing production of the voluminous quantities of these hormones during human pregnancy.     

PAL31 expression in rat trophoblast giant cells

PAL31 is a proliferation-related acidic nuclear protein that belongs to the leucine-rich protein family and is expressed cell-cycle-dependently. Trophoblasts differentiate into the trophoblast giant cells (TGCs) through the unusual type of cell cycle, namely endoreduplication. In the present study, we investigated the spatiotemporal pattern of PAL31 expression in rat placenta and Rcho-1 cell line. The PAL31 mRNA concentration varied in different areas of the placenta, and was barely detectable in the TGC layer. In Rcho-1 cells, although the level of PAL31 mRNA decreased dramatically during differentiation, PAL31 was detected even after differentiation. The site of intranuclear localization of PAL31 mostly overlapped with that of PCNA in the undifferentiated Rcho-1 cells, while they were not overlapped in differentiated cells. Thus, the subcellular localization of PAL31 in Rcho-1 cells significantly changed, and loss of cell cycle dependency after differentiation was noted. PAL31 is suggested to play a role in the endoreduplication distinct from the usual DNA duplication.