Review: the transcriptional and signalling networks of mouse trophoblast stem cells.

Trophoblast stem cells (TSCs) are a self-renewing stem cell population derived from the early trophoblast lineage, analogous to embryonic stem cells (ESCs) that can be generated from the inner cell mass (ICM) of the mouse blastocyst. In that sense TSCs and ESCs reflect the earliest lineage differentiation event after fertilization. TSCs are characterized by an indefinite proliferation potential and by multipotency, i.e. the ability to differentiate into all the various trophoblast cell types of the placenta. These properties are driven by specific signalling pathways orchestrating characteristic transcriptional outputs. Here we review the recent advances in studying the signalling cascades and the transcriptional regulatory networks that define specification and maintenance of TSCs, and provide a future outlook of TSC research.

Regulation of early trophoblast differentiation - lessons from the mouse.

The earliest stages of trophoblast differentiation are of tremendous importance to mediate implantation and to lay the anatomical foundations for normal placental development and function throughout gestation. Yet our molecular insights into these early developmental processes in humans have been limited by the inaccessibility of material and the unavailability of trophoblast cell lines that fully recapitulate the behaviour of early placental trophoblast. In this review we highlight recent advances that have come from the study of distinct stem cell types representative of the embryonic and extraembryonic lineages in the mouse, and from the study of mouse mutants. These models have revealed the presence of intricate transcriptional networks that are set up by signalling pathways, translating extracellular growth factor and cell positional information into distinct lineage-specific transcriptional programmes. The trophoblast specificity of these networks is ensured by epigenetic mechanisms including DNA methylation and histone modifications that complement each other to define trophoblast cell fate and differentiation. Despite the anatomical differences between mouse and human placentas, it seems that important aspects of early trophoblast specification are conserved between both species. Thus we may be able to build on our insights from the mouse to better understand early trophoblast differentiation in the human conceptus which is important for improving assisted reproductive technologies and may enable us in the future to derive human trophoblast stem cell lines. These advances will facilitate the investigation of genetic, epigenetic and environmental influences on early trophoblast differentiation in normal as well as in pathological conditions.

Stem cells from fetal membranes - a workshop report.

Stem cells that can be derived from fetal membranes represent an exciting field of research that bears tremendous potential for developmental biology and regenerative medicine. In this report we summarize contributions to a workshop in which newest insights into the characteristics, subtypes and molecular determinants of stem cells from trophoblast and endometrial tissues were presented.

IFPA award in placentology lecture - characteristics and significance of trophoblast giant cells.

Extraembryonic development in rodents depends on the differentiation and function of trophoblast giant cells. Morphologically striking, giant cells exhibit many extraordinary characteristics adapted to ensure the success of pregnancy. This review summarizes some of the intriguing aspects of giant cell morphology and function. Giant cells are highly polyploid as a result of a switch from a mitotic to an endoreduplicative cell cycle. They further partition their genome content into various fragments which may represent a mechanism to maximize protein synthesis. Similar to metastatic tumour cells, they breach basement membranes and invade deeply into a foreign tissue, the maternal decidualized uterine stroma. Their angiogenic and vasodilatory properties, combined with the ability to remodel arterial walls, enable them to redirect maternal blood flow towards the implantation site. Recent advances have recognized that the giant cell population is more diverse than previously recognized and future studies will have to show how these subtypes differ functionally and how their differentiation is controlled.

Epigenetic landscape required for placental development.

Formation of extraembryonic tissues, and in particular the placenta, is an absolute necessity to ensure growth and survival of the embryo during intrauterine development in mammals. To date, an intriguing number of genes have been identified that are essential for development of extraembryonic structures. However, the underlying genetic information must be interpreted by a set of epigenetic instructions to both establish and maintain lineage- and cell type-specific expression profiles. Based on accumulating data in particular from studies in the mouse, this article is aimed at highlighting the epigenetic machinery required for differentiation of extraembryonic cell types and formation of the placenta. An overview of knockout models reveals key stages in extraembryonic development that are particularly sensitive to alterations in the chromatin environment. The article also summarizes the importance of complex epigenetically controlled mechanisms for placental development, such as imprinted gene expression and imprinted X chromosome inactivation. These investigations of the epigenetic regulation of transcriptional states will provide valuable insights into the dynamic chromatin environment that is specific to extraembryonic tissues and determines gene expression patterns required for normal trophoblast differentiation.

Epigenetics and imprinting of the trophoblast -- a workshop report.

Genomic imprinting is a remarkable process that causes genes to be expressed or repressed depending on their parental-origin. Imprinted genes play important roles in prenatal growth and organ development. Postnatally, imprinted genes can contribute to the regulation of metabolic pathways and behaviour associated with the control of resources. One of the most important sites of imprinted gene action is the placenta. During this workshop at the 11th meeting of the International Federation of Placenta Associations/European Placenta Group held in Glasgow, a series of short talks were presented providing an overview of the evolution, function and mechanisms of imprinting in mammals with particular reference to the placenta. In addition, epigenetic control of trophoblast development and function were considered. This report summarises the contributions to the workshop.

Genetic and genomic approaches to study placental development.

Recent technological advances in genetic manipulation and expression profiling offer excellent opportunities to elucidate the molecular mechanisms controlling developmental processes during embryogenesis. Thus, this revolution also strongly benefits studies of the molecular genetics of placental development. Here we review the findings of several expression profiling analyses in extraembryonic tissues and assess how this work can contribute to the identification of essential components governing placental development. We further discuss the relevance of these components in the context of genetic manipulation experiments. In conclusion, the intelligent combination of genetic and genomic approaches will substantially accelerate the progress in identifying the key molecular pathways of placental development.

Divergent genetic and epigenetic post-zygotic isolation mechanisms in Mus and Peromyscus.

Interspecific hybridization in the rodent genera Peromyscus and Mus results in abnormal placentation. In the Peromyscus interspecies hybrids, abnormal allelic interaction between an X-linked locus and the imprinted paternally expressed Peg3 locus was shown to cause the placental defects. In addition, loss-of-imprinting (LOI) of Peg3 was positively correlated with increased placental size. As in extreme cases this placental dysplasia constitutes a post-zygotic barrier against interspecies hybridization, this finding was the first direct proof that imprinted genes may be important in speciation and thus in evolution. In the Mus interspecies hybrids, a strong role of an X-linked locus in placental dysplasia has also been detected. However, here we show by backcross and allele specific expression analyses that neither LOI of Peg3 nor abnormal interactions between Peg3 and an X-linked locus are involved in generating placental dysplasia in Mus hybrids, although the placental phenotypes observed in the two genera seem to be identical. In contrast to this, another dysgenesis effect common to Peromyscus and Mus hybrids, altered foetal growth, is caused at least in part by the same X-chromosomal regions in both genera. These findings first underline the strong involvement of the X-chromosome in the genetics of speciation. Secondly, they indicate that disruption of epigenetic states, such as LOI, at specific loci may be involved in hybrid dysgenesis effects in one group, but not in another. Thus, we conclude that even in closely related groups divergent molecular mechanisms may be involved in the production of phenotypically similar post-zygotic barriers against hybridization.

Genes, development and evolution of the placenta.

Through studies of transgenic and mutant mice, it is possible to describe molecular pathways that control the development of all major trophoblast cell subtypes and structures of the placenta. For example, the proliferation of trophoblast stem cells is dependent on FGF signalling and downstream transcription factors Cdx2, Eomes and Err2. Several bHLH transcription factors regulate the progression from trophoblast stem cells to spongiotrophoblast and to trophoblast giant cells (Id1/2, Mash2, Hand1, Stra13). Intercellular actions critical for maintaining stable precursor cell populations are dependent on the gap junction protein Cx31 and the growth factor Nodal. Differentiation towards syncytiotrophoblast as well as the initiation of chorioallantoic (villous) morphogenesis is regulated by the Gcm1 transcription factor, and subsequent labyrinth development is dependent on Wnt, HGF and FGF signalling. These insights suggest that most of the genes that evolved to regulate placental development are either identical to ones used in other organ systems (e.g., FGF and epithelial branching morphogenesis), were co-opted to take on new functions (e.g., AP-2gamma, Dlx3, Hand1), or arose via gene duplication to take on a specialized placental function (e.g., Gcm1, Mash2). Many of the human orthologues of these critical genes show restricted expression patterns that are consistent with a conserved function. Such information is aiding the comparison of the human and mouse placenta. In addition, the prospect of a conserved function clearly suggests potential mechanisms for explaining complications of human placental development.

Trophoblast functions, angiogenesis and remodeling of the maternal vasculature in the placenta.

One of the most important local adaptations to pregnancy is the change in maternal blood flow to the implantation site. In rodents and primates, new blood vessels form through angiogenesis, dilate and then become modified such that the blood enters into trophoblast cell-lined sinuses (hemochorial). Evidence from gene knockout mice suggests that factors from the placenta regulate the uterine vasculature. Consistent with this, trophoblast giant cells produce a number of angiogenic and vasoactive substances that may mediate these effects. Teratocarcinomas containing large numbers of trophoblast giant cells (derived from Parp1 gene-deficient ES cells) show similar 'hemochorial' host blood flow, implying that the effects are not specific to the uterine vascular bed. As in primates, murine trophoblast cells also invade into the uterine arteries of the mother. However, in normal pregnancy, dilation of the uterine arteries may be largely mediated by the effect of uterine natural killer cells.

The role of the X chromosome in mammalian extra embryonic development.

Accumulating evidence points to the importance of the X chromosome for trophoblast development. In rodents, the extraembryonic cell lineage differs from somatic tissues in that X chromosome inactivation is imprinted, preferentially silencing the paternal X chromosome. As a consequence, trophoblast development is extremely susceptible to deviations from normal X inactivation and is impaired in situations of increased and reduced X-linked gene dosage. Mouse mutants have also shown that maintenance of X chromosome silencing in extraembryonic tissues requires a special set of heterochromatin proteins. Moreover, the X chromosome has been implicated in causing several malformations of the placenta. The observed importance of the X chromosome for placental development can be explained by the presence of many trophoblast-expressed genes, especially in the proximal and central regions. Given that the placenta represents a postzygotic barrier to reproduction, evolutionary constraints may be responsible for the presence of placental genes on the X chromosome that are often co-expressed in brain and testis.

UniGene cDNA array-based monitoring of transcriptome changes during mouse placental development.

The placenta is a highly specialized organ essential for embryonic growth and development. Here, we have applied cDNA subtraction between extraembryonic tissues of early- (day 7.5 of gestation) and late-stage embryos (day 17.5) to generate stage-specific cDNA pools that were used for screening of high-density mouse UniGene cDNA arrays containing 25,000 clones. A total of 638 clones were identified, 488 with the e7.5-specific probe and 150 with the e17.5-specific probe. Importantly, 363/638 (56.9%) of the hybridizing clones were not known to be expressed during placental development before. Differential regulation was confirmed by Northern blot and in situ hybridization for a total of 44/44 of positive clones. Thus, this combination of cDNA subtraction and array hybridization was highly successful for identification of genes expressed and regulated during placental development. These included growth factors and receptors, components of the transcriptional and translational machinery, cell cycle regulators, molecular chaperones, and cytoskeletal elements. The extensive in situ hybridization analysis revealed extraembryonic structures with a high density of differentially expressed genes, most strikingly the ectoplacental cone and the spongiotrophoblast. This large-scale identification of genes regulated during placentogenesis is extremely useful to further elucidate the molecular basis of extraembryonic development.

Genes governing placental development.

The placenta is essential for fetal growth because it promotes the delivery of nutrients and oxygen from the maternal circulation. In mice, many gene mutations disrupt formation of the placenta, with specific effects at different times and on different components. Studies of these mutations are beginning to provide insights into both the molecular pathways required for formation of different placental substructures and the nature of intercellular interactions, between trophoblast, mesenchymal and vascular components, that regulate placental development. Conserved gene expression patterns in humans should enable the elucidation of the molecular basis of human placental dysfunction.

Genetic and developmental analysis of X-inactivation in interspecific hybrid mice suggests a role for the Y chromosome in placental dysplasia.

It has been shown previously that abnormal placental growth, i.e., hyper- and hypoplasia, occurs in crosses and backcrosses between different mouse (Mus) species. A locus that contributes to this abnormal development has been mapped to the X chromosome. Unexpectedly, an influence of fetal sex on placental development has been observed, in that placentas attached to male fetuses tended to exhibit a more pronounced phenotype than placentas attached to females. Here, we have analyzed this sex dependence in more detail. Our results show that differences between male and female placental weights are characteristic of interspecific matings and are not observed in intraspecific Mus musculus matings. The effect is retained in congenic lines that contain differing lengths of M. spretus-derived X chromosome. Expression of the X-linked gene Pgk1 from the maternal allele only and lack of overall activity of two paternally inherited X-linked transgenes indicate that reactivation or lack of inactivation of the paternal X chromosome in trophoblasts of interspecific hybrids is not a frequent occurrence. Thus, the difference between male and female placentas seems not to be caused by faulty preferential X-inactivation. Therefore, these data suggest that the sex difference of placental weights in interspecific hybrids is caused by interactions with the Y chromosome.

Semen collection, examination and spermiogram in ostriches.

The level of fertility in the male ostrich exerts considerable influence on the efficiency of the fertilization procedure, and thus also on reproductive performance. The determination of the reproductive capacity is of particular interest with regard to the selection of single individuals for optimizing reproduction ratios. Although the breeding and raising of ostriches has become increasingly important in many countries, little research has been completed on reproductive parameters and factors that may possibly influence them. This study presents observations made concerning the quantity and quality of sperm as found in the spermatological testing of 411 ejaculate samples taken from male ostriches on two farms in Namibia. The semen volume varied between 0.1 and 1.5 ml (mean, 0.64 ml). Normal ejaculate colours ranged from white to ivory; the consistency ranged from thin creamy to viscous. The measured pH values lay between 6.4 and 8.0 (mean, 7.3). Microscopic investigations revealed sperm concentrations of 8.9-78.1 million/microl and individual sperm motility from 42 to 96% (mean, 78%). No mass motility was detectable in 42% of the ejaculates; weak mass motility was found in 46%, and clear mass movements were to be found in only 12% of samples. Regarding the morphology of the sperm, 5 to 26% were abnormal (mean, 17%) and 4 to 28% (mean, 20%) were dead. Seasonal patterns of sperm concentration and the influence of frequency of semen collection were investigated in a group of 56 healthy male ostriches. Peak sperm concentrations were found at the beginning of the breeding season in spring; the lowest values were found at the end of the breeding season in autumn. The highest quality ejaculate was obtained from those males whose semen was collected once a week. The results of this study provide fundamental data for the establishment of minimum quality requirements for ostrich sperm to be met by individuals receiving certification as breeding animals and for the selection of suitable males for use in artificial insemination.

cDNA subtraction cloning reveals novel genes whose temporal and spatial expression indicates association with trophoblast invasion.

Trophoblast invasion is a critical process in development of most mammals that shares similarities with the invasive behavior of tumor cells. In the present investigation, a cDNA subtraction library was constructed between invasive trophoblast at day 8 of murine development and mature noninvasive placenta at day 18 of gestation. One of the differentially expressed clones, Epcs26, was mapped to the X chromosome and revealed no homology to any known gene. It was predominantly expressed in parietal endoderm, undifferentiated cells of the ectoplacental cone, and a few trophoblast giant cells. Another gene, designated Epcs50, was mapped to chromosome 19. It exhibited homologies to the mouse Mps1 gene and, like Mps1, may have a distant relationship to the lytic protein perforin. High expression was detected in parietal endoderm cells and in a subset of secondary trophoblast giant cells. Two sequences, Epcs24 and Epcs68, exhibited an extensive open reading frame that shared the common features of the cysteine proteinase cathepsin L. Expression was confined to an undefined subpopulation of trophoblast giant cells. Both genes were mapped to chromosome 13 in close proximity to cathepsins L and J. The known functions of MPS1 and cathepsin L proteins indicate that the related proteins EPCS50, EPCS24, and EPCS68 participate in conferring invasive properties to the mouse trophoblast.

Silencing of the Y-chromosomal gene tspy during murine evolution.

We have studied the process of tspy gene silencing in murine evolution. We have isolated functional tspy sequences from Apodemus agrarius, A. sylvaticus, A. flavicollis, and Mus platythrix (subgenus Pyromys) and nonfunctional tspy sequences from species of the subgenus Mus. We present two alternative models as to how tspy may have lost its function in the murine lineage.

Expression of the imprinted genes MEST/Mest in human and murine placenta suggests a role in angiogenesis.

In the mouse fetus, Mest is widely expressed in mesoderm derived tissues. In separate studies in mice and in humans, it has been shown to be maternally imprinted, that is, only the paternally inherited allele is active. Here, we show that starting with implantation, Mest is also expressed in maternal decidua of the mouse and in placenta of both humans and mice. Expression in murine decidua was restricted to endothelial cells. After Day 7, expression in the decidua gradually decreased. Mest-specific RT-PCR and restriction fragment length variant (RFLV) analysis of decidualized endometrium isolated from (M. musculus x M. spretus)F1 females showed that only the paternally derived Mest allele was activated in the decidual endothelium. In the mouse extraembryonic tissues, Mest transcripts were detected in derivatives of extraembryonic mesoderm only. Here, hemangioblast precursor cells and endothelial cells were positive. At all developmental stages of the mouse, trophoblast-derived cells were clearly devoid of Mest transcripts. In the human placenta MEST transcripts were also detected in hemangioblast precursor cells, however, MEST was also expressed in villous and invasive cytotrophoblast. In a human choriocarcinoma/trophoblastic tumour grown in a nude mouse, human MEST was expressed in the tumour cells, whereas murine Mest was expressed in endothelia of the murine capillaries. The expression pattern exhibited by both Mest and MEST in extraembryonic tissues during development and during formation of choriocarcinoma/trophoblast tumour suggests a functional role of the MEST proteins related to oncofetal angiogenesis. Dev Dyn 2000;217:1-10.