Rescue of keratin 18/19 doubly deficient mice using aggregation with tetraploid embryos

We have previously shown that the targeted deletions of both type I keratins (K) 18 and 19 cause lethality by embryonic day (e) 9.5 due to fragility and cytolysis of trophoblast giant cells. The development of the embryo proper appeared to be unaffected and its death was caused by nutrient deficiency. In order to address the function of keratins within the embryo proper, lethality due to extraembryonic tissue failure must be overcome. One approach to rescue doubly deficient embryos is by aggregating knockout embryos with tetraploid wild-type embryos. As a general tool, tetraploid aggregation can be used to rescue embryonic lethality caused by defects in extraembryonic tissues like the placenta, trophoblast or yolk sac. We rescued K18-/- K19-/- embryos until e11.5, using this approach, proving that the loss of the keratin cytoskeleton causes defects in the trophoblast giant cell layer, but has no effect on early development of the embryo proper.     

Cytokeratins 8 and 19 in the mouse placental development

To investigate the expression and biological roles of cytokeratin 19 (K19) in development and in adult tissues, we inactivated the mouse K19 gene (Krt1-19) by inserting a bacterial beta-galactosidase gene (lacZ) by homologous recombination in embryonic stem cells, and established germ line mutant mice. Both heterozygous and homozygous mutant mice were viable, fertile, and appeared normal. By 7.5-8.0 days post coitum (dpc), heterozygous mutant embryos expressed lacZ in the notochordal plate and hindgut diverticulum, reflecting the fact that the notochord and the gut endoderm are derived from the axial mesoderm-originated cells. In the adult mutant, lacZ was expressed mainly in epithelial tissues. To investigate the possible functional cooperation and synergy between K19 and K8, we then constructed compound homozygous mutants, whose embryos died approximately 10 dpc. The lethality resulted from defects in the placenta where both K19 and K8 are normally expressed. As early as 9. 5 dpc, the compound mutant placenta had an excessive number of giant trophoblasts, but lacked proper labyrinthine trophoblast or spongiotrophoblast development, which apparently caused flooding of the maternal blood into the embryonic placenta. These results indicate that K19 and K8 cooperate in ensuring the normal development of placental tissues.     

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.     

Cathepsin proteases have distinct roles in trophoblast function and vascular remodelling

Trophoblast giant cells are instrumental in promoting blood flow towards the mouse embryo by invading the uterine endometrium and remodelling the maternal vasculature. This process involves the degradation of the perivascular smooth muscle layer and the displacement of vascular endothelial cells to form trophoblast-lined blood sinuses. How this vascular remodelling is achieved at the molecular level remains largely elusive. Here, we show that two placenta-specific cathepsins, Cts7 and Cts8, are expressed in distinct but largely overlapping subsets of giant cells that are in direct contact with maternal arteries. We find that Cts8, but not Cts7, has the capacity to mediate loss of smooth muscle alpha-actin and to disintegrate blood vessels. Consequently, conditional ubiquitous overexpression of Cts8 leads to midgestational embryonic lethality caused by severe vascularization defects. In addition, both cathepsins determine trophoblast cell fate by inhibiting the self-renewing capacity of trophoblast stem cells when overexpressed in vitro. Similarly, transgenic overexpression of Cts7 and Cts8 affects trophoblast proliferation and differentiation by prolonging mitotic cell cycle progression and promoting giant cell differentiation, respectively. We also show that the cell cycle effect is directly caused by some proportion of CTS7 localizing to the nucleus, highlighting the emerging functional diversity of these typically lysosomal proteases in distinct intracellular compartments. Our findings provide evidence for the highly specialized functions of closely related cysteine cathepsin proteases in extra-embryonic development, and reinforce their importance for a successful outcome of pregnancy.     

Functional analysis of trophoblast giant cells in parthenogenetic mouse embryos

Diploid mouse embryos containing only maternal DNA (parthenotes) fail, in part, because the inner cell mass does not induce the trophoblast to grow. In this study, we asked whether any of the defects in parthenotes may arise from alterations in trophoblast function. We examined the expression of genes important for normal trophoblast function and found several trophoblast genes that were expressed at normal levels in the primary trophoblast cells of parthenotes: E-cadherin, a cell adhesion molecule, was expressed normally in both the ICM and trophectoderm of parthenogenetic blastocysts and blastocyst outgrowths; the gene for Hxt, a basic helix-loop-helix factor that regulates trophoblast development, was expressed in both zygotic and parthenogenetic giant cells; placental lactogen-1, a hormone that is normally secreted by trophoblast giant cells, was expressed in most of both parthenogenetic and normal trophoblast cells; and the 92 kDa matrix metalloproteinase, gelatinase B, also known as MMP-9, was secreted at equivalent levels by both zygotic and parthenogenetic blastocyst outgrowths. However, once the outgrowths had developed, a subpopulation of trophoblast cells in parthenogenetic embryos had decreased DNA replication and significantly fewer nucleoli per nucleus than did zygotic embryos. Moreover, the parthenogenetic trophoblast cells growing out from blastocysts had a decreased viability in culture. These data suggest that, although parthenogenetic embryos are able to initiate primary trophoblast differentiation, the stability and continued differentiation of trophoblast giant cells may be abnormal. Our data support the hypothesis that the deficiency of secondary trophoblast giant cells may contribute to the decline of parthenogenetic embryos and suggest that the factors controlling this subset of trophoblast are distinct from those for primary trophoblast. Dev Genet 20:1–10, 1997. © 1997 Wiley-Liss, Inc.     

The thrombomodulin-protein C system is essential for the maintenance of pregnancy

Disruption of the mouse gene encoding the blood coagulation inhibitor thrombomodulin (Thbd) leads to embryonic lethality caused by an unknown defect in the placenta. We show that the abortion of thrombomodulin-deficient embryos is caused by tissue factor-initiated activation of the blood coagulation cascade at the feto-maternal interface. Activated coagulation factors induce cell death and growth inhibition of placental trophoblast cells by two distinct mechanisms. The death of giant trophoblast cells is caused by conversion of the thrombin substrate fibrinogen to fibrin and subsequent formation of fibrin degradation products. In contrast, the growth arrest of trophoblast cells is not mediated by fibrin, but is a likely result of engagement of protease-activated receptors (PAR)-2 and PAR-4 by coagulation factors. These findings show a new function for the thrombomodulin-protein C system in controlling the growth and survival of trophoblast cells in the placenta. This function is essential for the maintenance of pregnancy.     

Ultrastructure of trophoblast giant cell transformation during the invasive stage of implantation of the mouse embryo

The fine structure of abembryonic and mural trophoblast cells of mouse embryos was analyzed during the initial stages of invasion of the endometrial stroma by the embryo (days 6-8 of pregnancy). On day 6 of pregnancy, most trophoblastic cells are flat and have spindle-shaped nuclei. A few large, round trophoblastic cells (giant cells) are present at the abembryonic pole. As pregnancy proceeds through days 7 and 8, the area occupied by the trophoblast becomes larger because of an increase in the trophoblastic cell population, growth of giant cells, and rearrangement of the latter cells into a network containing maternal blood. As flat cells transform into giant cells, their content of ribosomes, granular endoplasmic reticulum, Golgi complexes, lysosomelike bodies, and heterophagosomes increases. Reichert's membrane is always lined by cell bodies or by laminar processes of trophoblastic cells that are provided with small pores. Transformation of flat cells into giant cells is associated with an activation of the giant cells and their acquisition of invasive behavior.     

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-associated Kunitz-type serine proteinase inhibitor that was initially identified as a potent inhibitor of hepatocyte growth factor activator. HAI-1 is also a cognate inhibitor of matriptase, a membrane-associated serine proteinase. HAI-1 is expressed predominantly in epithelial cells in the human body. Its mRNA is also abundant in human placenta, with HAI-1 specifically expressed by villous cytotrophoblasts. In order to address the precise roles of HAI-1 in vivo, we generated HAI-1 mutant mice by homozygous recombination. Heterozygous HAI-1+/- mice underwent normal organ development. However, homozygous HAI-1-/- mice experienced embryonic lethality which became evident at embryonic day 10.5 postcoitum (E10.5). As early as E9.5, HAI-1-/- embryos showed growth retardation that did not reflect impaired cell proliferation but resulted instead from failed placental development and function. Histological analysis revealed severely impaired formation of the labyrinth layer, in contrast all other placental layers, such as the spongiotrophoblast layer and giant cell layer, which were formed. Our results indicate that mouse HAI-1 is essential for branching morphogenesis in the chorioallantoic placenta and lack of HAI-1 function may result in placental failure.     

Paracrine action of FGF4 during periimplantation development maintains trophectoderm and primitive endoderm

FGF4, a member of the fibroblast growth factor (FGF) family, is absolutely required for periimplantation mouse development, although its precise role at this stage remains unknown. The nature of the defect leading to postimplantation lethality of embryos lacking zygotic FGF4 is unclear and little is known about downstream targets of FGF4-initiated signaling within the various cellular compartments of the blastocyst. Here we report that postimplantation lethality of Fgf4(-/-) embryos is unlikely to reflect strictly mitogenic requirements for FGF4. Rather, our results suggest that FGF4 is required to maintain trophectoderm and primitive endoderm identity at embryonic day 4.5. This result is consistent with the reported in vitro activity of FGF4 in maintaining trophoblast stem cells and with the requirement for receptor tyrosine kinase signaling in primitive endoderm formation. Thus, postimplantation lethality of Fgf4(-/-) embryos likely results from the failure of proper differentiation and function of extraembryonic cell types.     

Targeted deletion of keratins 18 and 19 leads to trophoblast fragility and early embryonic lethality

It has been reported previously that keratin 8 (K8)-deficient mice of one strain die from a liver defect at around E12.5, while those of another strain suffer from colorectal hyperplasia. These findings have generated considerable confusion about the function of K8, K18 and K19 that are co-expressed in the mouse blastocyst and internal epithelia. To resolve this issue, we produced mice doubly deficient for K18 and K19 leading to complete loss of keratin filaments in early mouse development. These embryos died at around day E9.5 with 100% penetrance. The absence of keratins caused cytolysis restricted to trophoblast giant cells, followed by haematomas in the trophoblast layer. Up to that stage, embryonic development proceeded unaffected in the absence of keratin filaments. K18/19-deficient mouse embryos die earlier than any other intermediate filament knockouts reported so far, suggesting that keratins, in analogy to their well established role in epidermis, are essential for the integrity of a specialized embryonic epithelium. Our data also offer a rationale to explore the involvement of keratin mutations in early abortions during human pregnancies.     

Giant cell differentiation and lethality of homozygousYellow mouse embryos

The evidence presented herein strongly suggests that lethality of homozygousyellow embryos is expressed at the primary implantation stage of embryonic development. Furthermore the expression of the mutant gene appears to be restricted to a specific cell type—the trophoblast giant cell. The stage of developmental arrest is determined by the degree of differentiation and implantation of these cells. Receptivity of the endometrium to giant cell attachment is also an important contributing factor to lethality of homozygousA ^y embryos.     

Polyploidization dynamics of tertiary trophoblast giant cells in the rat placenta

Polyploidization peculiarities of tertiary giant trophoblast cells during their active detaching from the ectoplacental cone and migrating into decidua basalis are investigated. On the 12th day of gestation, the ploidy of the majority of cell nuclei varies within 4-8c, although there are a few 16c and 32c nuclei. On the 13th and 14th days of gestation, the ploidy level of tertiary giant trophoblast cells enhances; 8c and 16c nuclei prevail, the percentage of 32c nuclei increases, 64c nuclei arising. The ploidy level of tertiary giant cell coincides with the average and/or maximum ploidy degree of precursor cell populations. The significance of polyploidy as indispensable condition of differentiation of the trophoblast cells that actively invade into maternal tissues is discussed.     

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.     

A mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice

Mutations in genes encoding epidermal keratins cause skin disorders, while those in internal epithelial keratins, such as K8 and K18, are risk factors for liver diseases. The effect of dominant mutations in K8 or K18 during embryonic development and tissue homeostasis has not been examined so far. Here we demonstrate that the dominant mutation hK18 R89C, that is highly similar to hK14 R125C, causing EBS in humans, leads to cell type-specific lethality in mice, depending on the ratio of mutant to endogenous keratins. Mice expressing hK18 R89C in the absence of endogenous K19 and K18 died at mid-gestation from defects in trophoblast giant cells, accompanied by haematomas. A single, endogenous K18 allele rescued embryonic lethality but caused aggregation of keratins in all adult internal epithelia, surprisingly without spontaneous cell fragility. Closer analysis revealed that both filaments and aggregates coexisted in the same cell, depending on the ratio of mutant to endogenous keratins. Our results demonstrate that balanced overexpression of a wild-type keratin rescued the lethal consequences of a dominant-negative mutation. This has important implications for therapy approaches of keratinopathies, suggesting that suppressing the mutant allele is not necessary in vivo.