The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis.

The placenta and cardiovascular system are the first organ systems to form during mammalian embryogenesis. We show here that a single gene is critical for development of both. The Hand1 gene, previously called Hxt, eHAND and Thing1, encodes a basic helix-loop-helix (bHLH) transcription factor that starts to be expressed during pre-implantation development. After implantation, Hand1 expression is restricted to placental trophoblast cells and later to embryonic cardiac and neural crest cells. We generated Hand1-null mutant mice by gene targetting. Homozygous mutant embryos arrested by embryonic day (E) 7.5 of gestation with defects in trophoblast giant cell differentiation. This early mortality could be rescued by aggregation of mutant embryos with wild-type tetraploid embryos, which contribute wild-type cells to the trophoblast, but not the embryo. By E10.5, however, the Hand1-null fetuses derived from tetraploid chimaeras died due to cardiac failure. Their heart tubes showed abnormal looping and ventricular myocardial differentiation. Therefore, Hand1 is essential for differentiation of both trophoblast and cardiomyocytes, which are embryologically distinct cell lineages.

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.

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.

Implantation and the placenta: key pieces of the development puzzle.

The mammalian embryo cannot develop without the placenta. Its specialized cells (trophoblast, endoderm, and extraembryonic mesoderm) form early in development. They attach the embryo to the uterus (implantation) and form vascular connections necessary for nutrient transport. In addition, the placenta redirects maternal endocrine, immune, and metabolic functions to the embryo's advantage. These complex activities are sensitive to disruption, as shown by the high incidence of early embryonic mortality and pregnancy diseases in humans, as well as the numerous peri-implantation lethal mutations in mice. Integration of molecular and developmental approaches has recently produced insights into the molecules that control these processes.

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.

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.

Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E.

The stability of many proteins is controlled by the ubiquitin proteolytic system, which recognizes specific substrates through the action of E3 ubiquitin ligases [1]. The SCFs are a recently described class of ubiquitin ligase that target a number of cell cycle regulators and other proteins for degradation in both yeast and mammalian cells [2] [3] [4] [5] [6]. Each SCF complex is composed of the core protein subunits Skp1, Rbx1 and Cul1 (known as Cdc53 in yeast), and substrate-specific adaptor subunits called F-box proteins [2] [3] [4]. To understand the physiological role of SCF complexes in mammalian cells, we generated mice carrying a deletion in the Cul1 gene. Cul1(-/-) embryos arrested around embryonic day 6.5 (E6.5) before the onset of gastrulation. In all cells of the mutant embryos, cyclin E protein, but not mRNA, was highly elevated. Outgrowths of Cul1(-/-) blastocysts had limited proliferative capacity in vitro and accumulated cyclin E in all cells. Within Cul1(-/-) blastocyst cultures, trophoblast giant cells continued to endocycle despite the elevated cyclin E levels. These results suggest that cyclin E abundance is controlled by SCF activity, possibly through SCF-dependent degradation of cyclin E.

The HAND1 basic helix-loop-helix transcription factor regulates trophoblast differentiation via multiple mechanisms.

The basic helix-loop-helix (bHLH) transcription factor genes Hand1 and Mash2 are essential for placental development in mice. Hand1 promotes differentiation of trophoblast giant cells, whereas Mash2 is required for the maintenance of giant cell precursors, and its overexpression prevents giant cell differentiation. We found that Hand1 expression and Mash2 expression overlap in the ectoplacental cone and spongiotrophoblast, layers of the placenta that contain the giant cell precursors, indicating that the antagonistic activities of Hand1 and Mash2 must be coordinated. MASH2 and HAND1 both heterodimerize with E factors, bHLH proteins that are the DNA-binding partners for most class B bHLH factors and which are also expressed in the ectoplacental cone and spongiotrophoblast. In vitro, HAND1 could antagonize MASH2 function by competing for E-factor binding. However, the Hand1 mutant phenotype cannot be solely explained by ectopic activity of MASH2, as the Hand1 mutant phenotype was not altered by further mutation of Mash2. Interestingly, expression of E-factor genes (ITF2 and ALF1) was down-regulated in the trophoblast lineage prior to giant cell differentiation. Therefore, suppression of MASH2 function, required to allow giant cell differentiation, may occur in vivo by loss of its E-factor partner due to loss of its expression and/or competition from HAND1. In giant cells, where E-factor expression was not detected, HAND1 presumably associates with a different bHLH partner. This may account for the distinct functions of HAND1 in giant cells and their precursors. We conclude that development of the trophoblast lineage is regulated by the interacting functions of HAND1, MASH2, and their cofactors.

Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells.

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34(cdk1) complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Periodic expression of the cyclin-dependent kinase inhibitor p57(Kip2) in trophoblast giant cells defines a G2-like gap phase of the endocycle.

Endoreduplication is an unusual form of cell cycle in which rounds of DNA synthesis repeat in the absence of intervening mitoses. How G1/S cyclin-dependent kinase (Cdk) activity is regulated during the mammalian endocycle is poorly understood. We show here that expression of the G1/S Cdk inhibitor p57(Kip2) is induced coincidentally with the transition to the endocycle in trophoblast giant cells. Kip2 mRNA is constitutively expressed during subsequent endocycles, but the protein level fluctuates. In trophoblast giant cells synchronized for the first few endocycles, the p57(Kip2) protein accumulates only at the end of S-phase and then rapidly disappears a few hours before the onset of the next S-phase. The protein becomes stabilized by mutation of a C-terminal Cdk phosphorylation site. As a consequence, introduction of this stable form of p57(Kip2) into giant cells blocks S-phase entry. These data imply that p57(Kip2) is subject to phosphorylation-dependent turnover. Surprisingly, although this occurs in endoreduplicating giant cells, p57(Kip2) is stable when ectopically expressed in proliferating trophoblast cells, indicating that these cells lack the mechanism for protein targeting and/or degradation. These data show that the appearance of p57(Kip2) punctuates the completion of DNA replication, whereas its turnover is subsequently required to initiate the next round of endoreduplication in trophoblast giant cells. Cyclical expression of a Cdk inhibitor, by terminating G1/S Cdk activity, may help promote the resetting of DNA replication machinery.

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.

Unique features of the trophoblast interferons.

The trophoblast interferons (IFN) are Type I IFN with about 50% amino acid sequence identity to the leukocyte IFN (IFN-alpha). They are the major secretory products of the trophoblast of ruminant ungulate species during pregnancy in the period immediately preceding attachment and implantation when they have been implicated in the phenomenon known as maternal recognition of pregnancy. The trophoblast IFN have antiviral and antiproliferative activities typical of other Type I IFN, but unlike IFN-alpha, -beta and -omega are poorly responsive to viral induction and have a highly restricted pattern of expression. Nevertheless, a recombinant bovine IFN-alpha can mimic many of the properties of the trophoblast IFN and has been used pharmacologically to improve pregnancy success in sheep. It still remains unclear, however, whether the trophoblast IFN have unique biological properties or whether they are unusual merely by virtue of the location, magnitude and temporal nature of their expression at a critical time during pregnancy.

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.

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.

Multiple regulatory elements are required to direct trophoblast interferon gene expression in choriocarcinoma cells and trophectoderm.

Interferon-tau (IFN tau) is produced exclusively by the trophectoderm during the peri-implantation stage of pregnancy in ruminant ungulate species. Human choriocarcinoma cells (Jar) stably transfected with 1.8 kilobases of promoter from a bovine IFN tau gene ahead of a human GH (hGH) reporter gene constitutively synthesize hGH, but expression is not increased further by exposure to Newcastle disease virus. This and earlier experiments suggest that the transcriptional cues regulating IFN tau expression are distinct from those operating on other type I IFN genes. Transient transfection experiments reveal that two distinct promoter regions are required for full constitutive expression: one proximal (to position -126), which directs basal expression, and a more distal promoter region (positions -280 to -400), which acts as an enhancer. Nuclear extracts prepared from ovine conceptuses during the period of IFN tau expression interact with the proximal promoter region (positions -34 to -126) to form several complexes of high electrophoretic mobility. Although nucleotide sequence motifs potentially capable of binding the transcription factor IRF-1 are present in this region, IRF-1 does not transactivate the IFN tau gene. The distal part of the promoter contains only one region (-322 to -358) that forms a complex with these conceptus nuclear extracts. Both proximal and distal gel shift patterns become dramatically different when IFN tau gene expression ceases, perhaps reflecting the appearance of transcriptional repressors. Together these experiments support the conclusion that the control of IFN tau gene expression is very different from that of other type I IFN genes and that trophoblast-specific expression depends upon distal as well as proximal promoter regulatory elements.

The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon).

Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle. Here we describe production of bTP-1 by recombinant procedures in Escherichia coli. A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein. This construct was placed under the control of the Trp promoter within the expression vector pTrp2. Expression occurred optimally in E. coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium. The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein. The inclusion bodies were isolated by differential centrifugation and washed. The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air. Final purification was achieved by anion exchange chromatography on DEAE-cellulose. The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter. The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle. The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Activin is a local regulator of human cytotrophoblast cell differentiation.

Cytotrophoblast cells of the first trimester placenta are highly invasive when removed from villi and cultured in vitro. In vivo, however, only selected cytotrophoblast cells break through the overlying syncytiotrophoblast to form cytotrophoblast columns and ultimately invade the endometrium. To explore the role of paracrine growth factors in regulating cytotrophoblast development, we cultured explants of first trimester chorionic villi in vitro. Both activin and inhibin, as well as the activin binding protein follistatin, are produced by various trophoblast cells throughout pregnancy. We found that addition of activin-A, but not inhibin-A, stimulated the outgrowth of cytotrophoblast cells into the surrounding matrix. This outgrowth was characteristic of that observed in extravillous cytotrophoblast cells in vivo; it was accompanied by cell division within the proximal region of the cytotrophoblast outgrowth, synthesis of fibronectin, as well as the expression of markers characteristic of invasive cytotrophoblast cells, human leukocyte antigen-G and matrix metalloproteinase (MMP)-9. Activin also specifically induced the early expression of MMP-2 within villous cytotrophoblast cells. Addition of the activin binding protein, follistatin, blocked all of the effects of exogenous activin. The morphological and biochemical effects of activin were similar to those observed when signaling of endogenous transforming growth factor-beta was blocked. Interestingly, the latter effects were also reversed by the addition of follistatin. These data suggest that activin plays a local role in promoting cytotrophoblast column formation, likely by regulating the differentiation of villous cytotrophoblast into extravillous cytotrophoblast cells.

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.

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.