Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta

An increasing number of genes known to be critical for cell cycle control, differentiation, and tumor suppression have been found to impact development of the placenta. To elucidate how these genes contribute to development of embryonic and extra-embryonic lineages, we generated a transgenic mouse in which the Cre transgene is driven by placenta-specific regulatory sequences from the human CYP19 gene. Using ROSA26 conditional reporter mice, we could detect expression of the CYP19-Cre transgene throughout the extra-embryonic ectoderm and in the ectoplacental cone at embryonic day 6.5 (E6.5). By E11.5, recombination of LoxP reporter sites was detected in all derivatives of trophoblast stem cells, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. We conclude that the CYP19-Cre transgenic mouse developed here can be used in combination with conditional alleles to distinguish between embryonic and extra-embryonic gene function, and to begin to map the period of time when gene function is critical during development.     

Protein synthesis in embryonic tissues during mouse postimplantation development

Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (M(r)) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.     

Teratoid Tumors Derived from Mouse Embryos Grown in an Immunologically Privileged Site

Mouse early embryos and embryo fragments were transplanted into an immunologically privileged site, consisting of a glass cylinder previously implanted under the skin of adult mice in order to test their tumor producing potential, in allogeneic adult recipients. The highest yield of tumors was obtained upon transplantation of 6 1/2 day old embryos in toto. i.e., including the embryonic and extraembryonic areas. Histological examination showed teratomas composed of differentiated tissues derived from the three germ layers containing isolated foci of undifferentiated cells and nodules of trophoblast giant cells. Areas exhibiting the histological appearance of yolk sac carcinoma were also observed. Transplantation of the whole 6 1/2 day old egg cylinder, including the ectoplacental cone, and the isolated embryonic area produced a lower incidence of teratomas with a reduced variety of differentiated tissues. No yolk sac carcinoma was found in these grafts. The ectoplacental cone of 6 1/2 day embryos produced no tumors. Grafts of genital ridges from 12 1/2 day embryos gave rise to teratomas with well differentiated tissues of embryonic and extraembryonic origin. Areas ressembling yolk sac carcinoma were also observed. The life span of trophoblastic giant cells within the glass cylinder was significantly longer than in other experimental systems.     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Gene expression of cytokeratin endo A and endo B during embryogenesis and in adult tissues of mouse.

We have examined the pattern of gene expression of mouse cytokeratin endo A and endo B during postimplantational development and in adult organs by Northern blot and in situ hybridization analyses. Both mRNAs localized in the ectoplacental cone, trophoblastic giant cells surrounding the parietal yolk sac, trophoblast cells in placenta, visceral yolk sac, and simple epithelium of the embryo during postimplantational development and in simple or transitional epithelial tissues in adult organs. These results indicate that endo A and endo B are coexpressed and may play some roles in these tissues.     

Expression profiles of extracellular superoxide dismutase during mouse organogenesis

Although extracellular superoxide dismutase (EC-SOD), which scavenges the superoxide anion in extracellular spaces, has previously been implicated in the prenatal pulmonary response to oxidative stress in the developing lungs, little is currently known regarding the schematic expression pattern and the roles played by EC-SOD during embryogenesis. In an effort to characterize the pattern of EC-SOD expression during mouse organogenesis, quantitative RT-PCR, Western blotting, and in situ hybridization analyses were conducted in mouse embryos and extraembryonic tissues including placenta on embryonic days (Eds) 7.5-18.5. EC-SOD mRNA and protein were expressed in all the embryos and extraembryonic tissues examined. The mRNA level was higher in the embryos than the extraembryonic tissues on Eds 7.5-10.5, but after Ed 13.5, it evidenced an increasing pattern in the extraembryonic tissues. EC-SOD immunoreactivity also increased in the extraembryonic tissues after Ed 13.5. During organogenesis, EC-SOD mRNA was expressed principally in the ectoplacental cone, amnion, and neural ectoderm on Ed 7.5 and in the neural folds and primitive streak on Ed 8.5. On Eds 9.5-12.5, EC-SOD mRNA was expressed abundantly in the nervous tissues and forelimb and hindlimb buds. On Eds 13.5-18.5, EC-SOD mRNA was observed at high levels in the airway epithelium of lung, liver, the intestinal epithelium, skin, vibrissae, the metanephric corpuscle of kidney, the nasal cavity, and the labyrinth trophoblast, spongiotrophoblast, and blood cells in placenta. Our overall results indicate that EC-SOD is expressed spatiotemporally in developing embryos and surrounding extraembryonic tissues during mouse organogenesis, thus suggesting that EC-SOD may be relevant to organogenesis, playing the role of an antioxidant enzyme against endogenous and exogenous oxygen stresses.     

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.     

Yolk sac carcinoma derived from the rat epiblast as a renal isograft

We report the novel observation that a biphasic, parieto-visceral (PYS/VYS) yolk sac carcinoma can develop from the isolated epiblast of the pre-primitive streak rat embryo in a prolonged cultivation in vivo as a renal isograft. Late 7-day rat egg cylinders were dissected free of the ectoplacental cone and the Reichert's membrane. The middle segment of the cylinder, in which the embryonic and the extraembryonic cell layers partly overlap, were also removed. From the rest of the cylinder the 4 cell layers were isolated and transplanted separately under the kidney capsule of isogenic adult males. After 4 weeks the hypoblast was resorbed, the extraembryonic ectoderm gave rise to hemorrhagic cysts and trophoblastic giant cells, the extraembryonic (visceral yolk sac) endoderm formed benign cystic PYS/VYS tumors, and the epiblast developed into a benign teratoma. After prolonged (7-30 weeks) development of these teratomas as isografts, a malignant yolk sac carcinoma (YSC) developed in 45% of them. It destroyed the teratoma and the recipient's kidney, metastasized to peritoneum and other sites, and caused abundant ascites containing clustered tumor cells. The primary tumor was retransplantable subcutaneously as well as intraperitoneally, and displayed the characteristics of the mixed or biphasic PVYS carcinoma, with a progressive loss of the VYS component with time. Several data are apparently in favor of its origin by transdifferentiation rather than from undifferentiated cells.     

Differential gene expression during early murine yolk sac development

The visceral yolk sac (VYS), composed of extraembryonic mesoderm and visceral endoderm, is the initial site of blood cell development and serves important nutritive and absorptive functions. In the mouse, the visceral endoderm becomes a morphologically distinct tissue at the time of implantation (E4.5), while the extraembryonic mesoderm arises during gastrulation (E6.5–8.5). To isolate genes differentially expressed in the developing yolk sac, polymerase chain reaction (PCR) methods were used to construct cDNA from late primitive streak to neural plate stage (E7.5) murine VYS mesoderm and VYS endoderm tissues. Differential screening led to the identification of six VYS mesoderm-enriched clones: ribosomal protein L13a, the heat shock proteins hsc 70 and hsp 86, guanine-nucleotide binding protein-related gene, cellular nucleic acid binding protein, and ã-enolase. One VYS endoderm-specific cDNA was identified as apolipoprotein C2. In situ hybridization studies confirmed the differential expression of these genes in E7.5 yolk sac tissues. These results indicate that representative cDNA populations can be obtained from small numbers of cells and that PCR methodologies permit the study of gene expression during early mammalian postimplantation development. While all of the mesoderm-enriched genes were ubiquitously expressed in the embryo proper, apolipoprotein C2 expression was confined to the visceral endoderm. These results are consistent with the hypothesis that at E7.5, the yolk sac endoderm provides differentiated liver-like functions, while the newly developing extraembryonic mesoderm is still a largely undifferentiated tissue. © 1995 wiley-Liss, Inc.     

Development of trophoblast and placenta of the mouse. A reinvestigation with regard to the in vitro culture of mouse trophoblast and placenta

At 5 days post conceptionem (p.c.) shortly after implantation, giant cell transformation starts at the abembryonic pole of the blastocyst, spreading over the mural trophoblast; 1 day later, the first ectoplacental giant cells appear at the base of the fast growing ectoplacental cone (derived from the polar trophoblast). Giant cell transformation expands over it periphery. Thus, by the 8th day p.c., the conceptus is separated from the maternal tissue by a continuous layer of giant cells, variable in thickness. Giant cells reach their greatest size by 10 days p.c. in the mural tophoblast and by 12 days p.c. in the chorioallantoic placenta. They are probably no longer formed after that stage. Around the 8th day p.c., the allantois reaches contact with the ectoplacental cone, which develops into the chorioallantoic (definitive) placenta. At 9 days p.c., its four zones can already be discriminated: chorionic plate, labyrinth, junctional zone (trophospongium), and zone of giant cells, respectively. Within the next day, the chorioallantoic placental circulation is established. The yolk sac placental circulation is established by the 9th day p.c. The villi of the proximal layer of the yolk sac increase in size and number, and their capillary network becomes more dense until the 12th to 14th day p.c. This provides evidence that the yolk sac placenta exerts its function--to a certain extent--beyond the establishment of the definitive placenta. Around the 14th day p.c., the placental labyrinth reaches its definitive features. Fetal capillaries in the labyrinth, branching from unbilical blood vessels within the septa of connective tissue are surrounded by trophoblast cells. They form a dense vascular network bathing in maternal blood. The structures of the placental zones remain almost the same during further development, the borders becoming sometimes little blurred. Adjacent to the chorionic plate, subchorionic clefts appear at the 14th day p.c. These clefts become confluent to form the intraplacental space, regularly communicating with the yolk sac cavity. At the end of gestation (19th day p.c.) there is a considerable amount of eosinophilic material ('fibrinoid') between the zone of giant cells and the decidua, probably produced by the giant cells.     

Developmentally regulated expression of the cell-cell adhesion glycoprotein cell-CAM 120/80 in peri-implantation mouse embryos and extraembryonic membranes

Peri-implantation mouse embryos and extraembryonic membranes were examined immunohistochemically for the expression of the cell-cell adhesion molecule (cell-CAM) 120/80. Cell-CAM 120/80 was seen along the lateral borders of all cells in the blastocyst but became undetectable on trophoblastic giant cells, some mononuclear trophoblastic cells and parietal yolk sac cells when blastocysts were cultured in vitro. In postimplantation embryos in vivo, all parts of the early egg-cylinder reacted with the antibody to cell-CAM 120/80 except for the cells of the parietal endoderm and the primary trophoblastic giant cells. In the late stage egg-cylinder, no cell-CAM 120/80 was seen on the cells of the primitive mesoderm or on the primordial germ cells. The germ cells in genital ridges and fetal gonads remained cell-CAM 120/80-negative throughout the fetal stages of development. In the extraembryonic membranes, the visceral yolk sac, amnion, and the cells of the placental labyrinth were cell-CAM 120/80-positive, whereas, the parietal yolk sac cells and the spongiotrophoblast cells were negative. These data show that cell-CAM 120/80 is found on cells arranged into epithelial layers in the early embryo and extraembryonic tissues, but is not expressed in the dissociated cells differentiating from these epithelia. Thus, the expression of cell-CAM 120/80 appears to be developmentally regulated.     

Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)

Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.     

Origin and differentiation of the yolk sac and extraembryonic mesoderm in presomite human and rhesus monkey embryos.

Reexamination of presomite human and rhesus monkey embryos in the Carnegie Collection provides no evidence to corroborate the hypothesis that the trophoblast is the source of all extraembryonic tissues in these embryos. Instead, the present study indicates that the developmental pattern of the yolk sac and extraembryonic mesoderm is homologous to that in other eutharian mammals. The primary yolk sac of 10- to 11-day human blastocysts is partially filled with a meshwork of extraembryonic endoderm, whereas such a meshwork is absent in the rhesus monkey. It is suggested that this endodermal meshwork develops as the result of interstitial implantation in the human embryo. A small secondary yolk sac develops in 12- to 13-day human and macaque embryos as the result of pinching off of a portion of the larger primary yolk sac. Development of a secondary yolk sac in higher primates appears to be related causally to differential rates of expansion of the blastocyst and primary yolk sac within the simplex uterus. The caudal margin of the primitive streak develops precociously in 12- to 14-day human and macaque embryos, and this appears to be the source of all the extraembryonic mesoderm of the chorion, chorionic villi, and body stalk. It is suggested that the peripheral spread of extraembryonic mesoderm plays in inductive role in the development of chorionic villi, similar to other types of epithelial-mesenchymal inductive interactions. In contrast to previous hypotheses, the human and macaque trophoblasts appear to give rise only to additional trophoblast.     

The mouse KRAB zinc-finger protein CHATO is required in embryonic-derived tissues to control yolk sac and placenta morphogenesis

Yolk sac and placenta are required to sustain embryonic development in mammals, yet our understanding of the genes and processes that control morphogenesis of these extraembryonic tissues is still limited. The chato mutation disrupts ZFP568, a Krüppel-Associated-Box (KRAB) domain Zinc finger protein, and causes a unique set of extraembryonic malformations, including ruffling of the yolk sac membrane, defective extraembryonic mesoderm morphogenesis and vasculogenesis, failure to close the ectoplacental cavity, and incomplete placental development. Phenotypic analysis of chato embryos indicated that ZFP568 does not control proliferation or differentiation of extraembryonic lineages but rather regulates the morphogenetic events that shape extraembryonic tissues. Analysis of chimeric embryos showed that Zfp568 function is required in embryonic-derived lineages, including the extraembryonic mesoderm. Depleting Zfp568 affects the ability of extraembryonic mesoderm cells to migrate. However, explanted Zfp568 mutant cells could migrate properly when plated on appropriate extracellular matrix conditions. We show that expression of Fibronectin and Indian Hedgehog are reduced in chato mutant yolk sacs. These data suggest that ZFP568 controls the production of secreted factors required to promote morphogenesis of extraembryonic tissues. Our results support previously undescribed roles of the extraembryonic mesoderm in yolk sac morphogenesis and in the closure of the ectoplacental cavity and identify a novel role of ZFP568 in the development of extraembryonic tissues.     

Ultrastructure of the pre-implantation shark yolk sac placenta

During ontogeny, the yolk sac of viviparous sharks differentiates into a yolk sac placenta which functions in gas exchange and hematrophic nutrient transport. The pre-implantation yolk sac functions in respiration and yolk absorption. In a 10.0 cm embryo, the yolk sac consists of six layers, viz. (1) somatic ectoderm; (2) somatic mesoderm; (3) extraembryonic coelom; (4) capillaries; (5) endoderm; and (6) yolk syncytium. The epithelial ectoderm is a simple cuboidal epithelium possessing the normal complement of cytoplasmic organelles. The endoplasmic cisternae are dilated and vesicular. The epithelium rests upon a basal lamina below which is a collagenous stroma that contains dense bodies of varying diameter. They have a dense marginal zone, a less dense core, and a dense center. The squamous mesoderm has many pinocytotic caveolae. The capillary endothelium is adjacent to the mesoderm and is delimited by a basal lamina. The endoderm contains yolk degradation vesicles whose contents range from pale to dense. The yolk syncytium contains many morphologically diverse yolk granules in all phases of degradation. Concentric membrane lamellae form around yolk bodies as the main yolk granules begin to be degraded. During degradation, yolk platelets exhibit a vesicular configuration.     

Trophoblast-specific expression and function of the integrin alpha 7 subunit in the peri-implantation mouse embryo.

For implantation and placentation to occur, mouse embryo trophoblast cells must penetrate the uterine stroma to make contact with maternal blood vessels. A major component of the uterine epithelial basement membrane and underlying stromal matrix with which they interact is the extracellular matrix protein laminin. We have identified integrin alpha 7 beta 1 as a major receptor for trophoblast-laminin interactions during implantation and yolk sac placenta formation. It is first expressed by trophectoderm cells of the late blastocyst and by all trophectoderm descendants in the early postimplantation embryo through E8.5, then disappears except in cells at the interface between the allantois and the ectoplacental plate. Integrin alpha 7 expression is a general characteristic of the early differentiation stages of rodent trophoblast, given that two different cultured trophoblast cell lines also express this integrin. Trophoblast cells interact with at least three different laminin isoforms (laminins 1, 2/4, and 10/11) in the blastocyst and in the uterus at the time of implantation. Outgrowth assays using function-blocking antibodies show that alpha 7 beta 1 is the major trophoblast receptor for laminin 1 and a functional receptor for laminins 2/4 and 10/11. When trophoblast cells are cultured on substrates of these three laminins, they attach and spread on all three, but show decreased proliferation on laminin 1. These results show that the alpha 7 beta 1 integrin is expressed by trophoblast cells and acts as receptor for several isoforms of laminin during implantation. These interactions are not only important for trophoblast adhesion and spreading but may also play a role in regulating trophectoderm proliferation and differentiation.