Placental morphology: from molecule to mother -- a dedication to Peter Kaufmann -- a review.

Theories on the development of placental pathologies and insufficiencies are based on the assumption that the development of the placental villous trees, the invasion of extravillous trophoblast or the differentiation of the villous trophoblast is somehow dysregulated. One of the pioneers trying to answer these questions is Peter Kaufmann, who started research on the placenta nearly 40 years ago. In this review, we try to shed light on various aspects of placental development, and on how important morphology is in combination with other disciplines to elucidate the differences between a normal and a pathological placenta.

How to make a placenta: mechanisms of trophoblast cell differentiation in mice--a review.

The word placenta is derived from the Latin term meaning 'flat cake'. Despite the rather humble name, the placenta is an amazing organ that forms both the interface for selective delivery of nutrients from the mother to the fetus and also re-directs maternal metabolic, endocrine, cardiovascular and immune functions to promote fetal survival and growth. These two functions are fulfilled by different specialized trophoblast cell subtypes, and my laboratory has been studying how their formation and functions are regulated during placental development. Through molecular studies in cultured cells and tissues, genetic studies in mice, and comparative analysis of placentas from humans, rodents and farm animals, it is now possible to describe molecular pathways that control the development of all major trophoblast cell subtypes and structures of the placenta. The work has revealed an intricate complexity of cell-cell interactions, environmental factors, and molecular networks that control normal development.

Complex patterns of GCM1 mRNA and protein in villous and extravillous trophoblast cells of the human placenta.

The Gcm1 gene encodes a transcription factor that is essential for both syncytiotrophoblast differentiation and formation of chorionic villi in mice. Its early expression is very unusual in that it defines a subset of trophoblast cells in the chorion, a layer that otherwise contains trophoblast stem cells. While Gcm1 mRNA expression initiates independently within the chorion, the subsequent maintenance of mRNA expression as well as the onset of protein accumulation is dependent on contact with allantoic mesoderm. Previous studies have shown that human GCM1 mRNA and protein are detectable in the placenta, but their patterns have not been compared nor precisely localized. We, therefore, conducted the present study to determine if the human mRNA and protein are subject to the same complexities of regulation as the mouse. In situ hybridization studies showed that the GCM1 mRNA was expressed in villous cytotrophoblast cells, but only a subset and never within cells immediately at the base of columns. Interestingly, the mRNA was detected throughout the cytotrophoblast columns. GCM1 protein expression studies demonstrated that the transcription factor was present mainly within the nuclei of a subset of cytotrophoblast cells, consistent with its role as a transcription factor. Feint cytoplasmic staining of the transcription factor was found in the syncytiotrophoblast but not in aggregated syncytial nuclei. Nuclear immuno-reactivity for the GCM1 protein was detected in occasional nuclei in the distal part of the column. Therefore, GCM1 expression is regulated both at the transcriptional and translational level. Overall, these studies show that the general features of GCM1 mRNA and protein expression in the human placenta are conserved with the mouse. They also highlight the fact that villous cytotrophoblast cells are extremely heterogeneous with respect to GCM1 expression, a factor that should be considered when using isolated cytotrophoblast cells for culture studies.

The genetics of pre-eclampsia: a feto-placental or maternal problem?

Pre-eclampsia is a potentially life-threatening disease of women during pregnancy leading to hypertension and proteinuria. It affects 1 in 15 pregnancies but, despite intense research efforts, the cause of the disease remains mysterious. Because pre-eclampsia only occurs during pregnancy and its symptoms resolve after delivery, factors from the placenta are thought to be involved. The role of the placenta could be production of 'abnormal' factors that initiate widespread inflammation and vaso-constriction. Alternatively, because the placenta normally contributes to maternal cardiovascular adaptations of pregnancy, it may be that normal placental functions fail in pre-eclampsia or that susceptibilities in the mother to hypertensive, vascular and/or renal disease prevent the appropriate normal responses to them. The potential contributions of both maternal and fetal genes to the onset of the disease have complicated the genetic analysis of the disease in humans. Recent studies have identified strains of transgenic and mutant mice that develop the hallmark features of pre-eclampsia-like disease - gestational hypertension, proteinuria and kidney lesions (glomerulosclerosis). Comparison of three different mouse models suggests that pre-eclampsia can be initiated by at least three independent mechanisms: pre-existing borderline maternal hypertension that is exacerbated by pregnancy (BPH/5 strain of mice), elevated levels of the vasoconstrictor angiotensin II in the maternal circulation by placental over-production of renin (renin/angiotensinogen transgenic mice), and placental pathology (p57Kip2 mutant mice). These findings imply that the pathogenesis of pre-eclampsia cannot be explained by a single mechanism. Therefore, segregation of the human disease into different subtypes may be a key first step in identifying genetic risk factors.

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.

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.

Supporting the habit: income generation activities of frequent crack users compared with frequent users of other hard drugs.

US Federal sentencing guidelines punish possession of crack cocaine very differently from powder cocaine, based partially upon the assumption that crack users engage more frequently in criminal behavior to pay for their habit. This article analyzed frequent users (those who have used at least 15 of the last 30 days) of crack with subgroups of less frequent hard drug users in terms of various income generation activities reported during the past 30 days. The sample consists of 602 African-Americans who were current (in past 30 days) users or sellers of cocaine powder, crack, and heroin. They were carefully recruited from randomly selected blocks in the Central Harlem area of New York City and interviewed extensively in 1998-1999. Their IGAs were classified into six categories. Compared with not-frequent (less than 15 days) hard drug users, frequent crack and multiple hard drug users were equally likely to be involved in drug distribution activities, but were significantly less likely to have full-time jobs, part-time jobs, aid to families with dependent children or welfare support. They had much higher odds ratios for non-drug related illegal (theft mainly) income generation activities and sex work among women. Often, gender and birth cohort variables had higher odds ratios with specific income generation activities than the frequent use of the primary drug(s). This evidence suggests that very frequent crack users have been stigmatized by, are largely excluded from, and perform very marginal economic roles in the legal economic system (jobs and welfare), the illegal economic system, and even in the hard drug distribution system.

Imprinted X inactivation maintained by a mouse Polycomb group gene.

In mammals, dosage compensation of X-linked genes is achieved by the transcriptional silencing of one X chromosome in the female (reviewed in ref. 1). This process, called X inactivation, is usually random in the embryo proper. In marsupials and the extra-embryonic region of the mouse, however, X inactivation is imprinted: the paternal X chromosome is preferentially inactivated whereas the maternal X is always active. Having more than one active X chromosome is deleterious to extra-embryonic development in the mouse. Here we show that the gene eed (embryonic ectoderm development), a member of the mouse Polycomb group (Pc-G) of genes, is required for primary and secondary trophoblast giant cell development in female embryos. Results from mice carrying a paternally inherited X-linked green fluorescent protein (GFP) transgene implicate eed in the stable maintenance of imprinted X inactivation in extra-embryonic tissues. Based on the recent finding that the Eed protein interacts with histone deacetylases, we suggest that this maintenance activity involves hypoacetylation of the inactivated paternal X chromosome in the extra-embryonic tissues.

Placental development: lessons from mouse mutants.

The placenta is the first organ to form during mammalian embryogenesis. Problems in its formation and function underlie many aspects of early pregnancy loss and pregnancy complications in humans. Because the placenta is critical for survival, it is very sensitive to genetic disruption, as reflected by the ever-increasing list of targeted mouse mutations that cause placental defects. Recent studies of mouse mutants with disrupted placental development indicate that signalling interactions between the placental trophoblast and embryonic cells have a key role in placental morphogenesis. Furthering our understanding of mouse trophoblast development should provide novel insights into human placental function.

Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis.

BACKGROUND: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3'-kinase. By creating a null (Delta) allele of mouse Grb2, we have shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. RESULTS: Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. CONCLUSIONS: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways.

Late mitotic failure in mice lacking Sak, a polo-like kinase.

Polo-like kinases in yeast, flies, and mammals regulate key events in mitosis. Such events include spindle formation at G2/M, the anaphase-promoting complex (APC) at the exit from mitosis, the cleavage structure at cytokinesis, and DNA damage checkpoints in G2/M. Polo-like kinases are distinguished by two C-terminal polo box (pb) motifs, which localize the enzymes to mitotic structures. We previously identified Sak, a novel polo-like kinase found in Drosophila and mammals. Here, we demonstrate that the Sak kinase has a functional pb domain that localizes the enzyme to the nucleolus during G2, to the centrosomes in G2/M, and to the cleavage furrow during cytokinesis. To study the role of Sak in embryo development, we generated a Sak null allele, the first polo-like kinase to be mutated in mice. Sak(-/-) embryos arrested after gastrulation at E7.5, with a marked increase in mitotic and apoptotic cells. Sak(-/-) embryos displayed cells in late anaphase or telophase that continued to express cyclin B1 and phosphorylated histone H3. Our results suggest that Sak is required for the APC-dependent destruction of cyclin B1 and for exit from mitosis in the postgastrulation embryo.

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.

Genes regulating embryonic and fetal survival.

Embryonic mortality in both farm animals and humans occurs most frequently during the first few weeks after conception. It can be attributed to abnormalities in the earliest developmental processes during embryogenesis that include implantation, maternal recognition of pregnancy, and formation of the placenta and cardiovascular system. The molecular mechanisms that are essential for all of these early processes are being elucidated at a rapid pace using transgenic and gene knockout approaches in mice. Two important general conclusions have emerged from this work. First, placental defects can occur by a number of different molecular mechanisms and can result from defects in the development or function of its trophoblast, mesenchymal or vascular components. Second, placental and cardiovascular functions are intimately linked. Cells of the placenta, for example, produce hormones that have profound effects on maternal and fetal cardiac and vascular function. In addition, development of the two is linked mechanistically through the use of some genes that are essential for development of both. Understanding the molecular basis of these processes should help to address the major limits to the success of embryo transfer, IVF and embryo cloning practices in livestock species.

Early exclusion of hand1-deficient cells from distinct regions of the left ventricular myocardium in chimeric mouse embryos.

The basic helix-loop-helix transcription factor gene Hand1 has been implicated in development of the heart. However, the early lethality of Hand1-null mutant mouse embryos has precluded a precise understanding of its function. In this study, we have generated Hand1 homozygous mutant ES cells and performed in vitro differentiation experiments and chimeric analysis to study the role of Hand1 function during cardiac development. Hand1-null ES cells were able to differentiate into beating cardiomyocytes in vitro that expressed cardiac myosin and several cardiac-specific transcripts including Nkx2-5, alpha-cardiac actin, and the myofilament genes myosin light chain 2a and 2v. In chimeras derived from Hand1-null ES cells and ROSA26 embryos, mutant cells were underrepresented in the left caudal region of the linear heart tube at E8.0. By E9.5, after cardiac looping, mutant cells were underrepresented in the anterior region of the outer curvature of the left ventricular myocardium, but did contribute to other parts of the left ventricle and to other cardiac chambers. These results imply that Hand1 is not essential for differentiation of ventricular cardiomyocytes. Hand1-null cells were also underrepresented in several other regions of later embryos, including the rhombencephalic neural tube that was associated with a deficiency of mutant cells in the neural crest cell-derived cardiac outflow tract and first branchial arch. In summary, Hand1 has cell-autonomous functions during cardiac morphogenesis in both mesodermal and neural crest derivatives.

The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta.

Trophoblast cells of the placenta are established at the blastocyst stage and differentiate into specialized subtypes after implantation. In mice, the outer layer of the placenta consists of trophoblast giant cells that invade the uterus and promote maternal blood flow to the implantation site by producing cytokines with angiogenic and vasodilatory actions. The innermost layer, called the labyrinth, consists of branched villi that provide a large surface area for nutrient transport and are composed of trophoblast cells and underlying mesodermal cells derived from the allantois. The chorioallantoic villi develop after embryonic day (E) 8.5 through extensive folding and branching of an initially flat sheet of trophoblast cells, the chorionic plate, in response to contact with the allantois. We show here that Gcm1, encoding the transcription factor glial cells missing-1 (Gcm1), is expressed in small clusters of chorionic trophoblast cells at the flat chorionic plate stage and at sites of chorioallantoic folding and extension when morphogenesis begins. Mutation of Gcm1 in mice causes a complete block to branching of the chorioallantoic interface, resulting in embryonic mortality by E10 due to the absence of the placental labyrinth. In addition, chorionic trophoblast cells in Gcm1-deficient placentas do not fuse to form syncytiotrophoblast. Abnormal development of placental villi is frequently associated with fetal death and intrauterine growth restriction in humans, and our studies provide the earliest molecular insight into this aspect of placental development.

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.