Giant Cell Layer Research Articles

Lessons on Life from SENP2

Embryonic development is a wonderful thing to behold. Consider the creation of a human embryo: a single fertilized egg cell gives rise to an adult organism consisting of approximately 6 × 1013 cells, organized into myriad tissues. This amazing feat is achieved through a carefully choreographed developmental program, where the cells of the growing embryo make a series of cell fate decisions that guide their differentiation into specialized tissues. The first embryonic cell fate decision comes early after fertilization, when an embryo's cells differentiate into two distinct populations: the inner cell mass and the outer trophectoderm. The inner cell mass will go on to become the embryo proper, while trophectoderm cells are stem cells that will become the placenta, which nourishes and protects the developing organism. While the genes and pathways contributing to embryonic development have enjoyed close scientific scrutiny, very little is known about how the trophectoderm generates placental tissues. In this issue of PLoS Biology, Shang-Yi Chiu, Wei Hsu, and colleagues uncover a pathway that is important for the generation of trophectoderm-derived tissues. The murine placenta comprises three layers: the inner labyrinth layer, which filters the maternal blood supply for nutrients that can be absorbed for the embryo; the spongiotrophoblast layer, which physically supports the labyrinth layer; and an outer layer of trophoblast giant cells (TGC), which completely surrounds the embryo. The TGC cells are unique, because they have multiple copies of the genome (known as polyploid cells). However, when abnormal polyploidy (known as aneuploidy) occurs, it is associated with cancer. Several control mechanisms exist to prevent polyploidy; a prominent one is the tumor suppressor p53, which acts to halt cell growth in the presence of DNA damage or other forms of cellular stress. However, since the generation of polyploid cells in the placenta is part of the normal developmental program, some mechanism must allow TGC cells to evade the controls that prevent polyploidy in other tissues. Chiu and colleagues were led to examining placental development by their studies on a protein called SENP2, which is a member of a family of proteases that removes a small protein-tag called SUMO (short for “small ubiquitin-related modifier”) from other proteins. SUMO modifies the function and subcellular location of proteins to which it is attached, and SENP2 helps reverse these changes. While studying the role of SENP2 in mouse embryonic development, the researchers observed that SENP2 is specifically expressed in trophectoderm-derived tissues, and that embryos lacking SENP2 failed to properly develop any of the three placental tissue layers. This failure occurred because the cells that give rise to the placental tissues had undergone cell cycle arrest and were trapped in a state of suspended growth. The authors therefore set out to find SENP2 target proteins that could be involved in arresting cell growth. In the journey of their searches, they discovered that p53—or proteins that modify p53 activity—were affected by the SENP2 deficiency. Although p53 can be modified by SUMO, the group found that SENP2 indirectly regulates p53 activity in trophectoderm-derived tissues by modifying the subcellular location of another protein called Mdm2. Mdm2 is known to interact with p53 and induce p53 degradation. Mdm2 can undergo SUMO modification, and Chiu and colleagues found that SUMO-modified Mdm2 is found only in the cell nucleus, not the cytoplasm. Cells lacking SENP2 are deficient for removing SUMO from Mdm2, so Mdm2 becomes trapped in the nucleus, where it is unable to promote p53 degradation. This allows p53 to accumulate within the cells, and causes cell growth arrest. These effects were reversible by knocking down p53 expression by RNA interference. Furthermore, stimulation of p53 by blocking Mdm2 with a drug results in defects resembling the loss of SENP2. These findings have implications for both embryonic development and cancer. The work highlights a role for p53 in preventing polyploidy, because p53 repression is required for the polyploid trophoblast cells to properly develop; the study of trophectoderm-derived tissues could therefore provide further insights into the pathways that cells normally use to control polyploidy. The authors accordingly plan further studies to elucidate how SENP2-mediated regulation of p53 contributes to both normal and pathological cell growth processes. Placenta for cancer research: What do we learn from the first communication relay with our mothers? New and old players add to our understanding of cells with specialized machinery for genome replication.

Cervical ectopic pregnancy on the portio: conservative case management and clinical review

To present a case and management of an early ectopic pregnancy on the portio. Case study and literature review. Hospital outpatient clinic. A 38-year-old woman who presented in the outpatient clinic with a 2-week history of painless acyclic vaginal bleeding. Excision of the ectopic pregnancy under local anesthesia after clinical examination, urine pregnancy test, and serum ss-hCG measurement. Conservative treatment options and preservation of patient's reproductive capacity. Serum ss-hCG was raised. Complete excision of the ectopic lesion was performed without the need for administration of chemotherapeutic agents and curettage. Histology revealed fragments of the cervical wall with a layer of chorionic giant cells and one intact chorionic villi. Cervical pregnancy is a rare form of ectopic pregnancy. It can be associated with high morbidity and adverse consequences for future fertility, but spontaneous abortion is also possible. We present a case and successful management of an early ectopic pregnancy on the surface of the portio.

HOP/NECC1, A Novel Regulator of Mouse Trophoblast Differentiation

Homeodomain-only protein/not expressed in choriocarcinoma clone 1 (HOP/NECC1) is a newly identified gene that modifies the expression of cardiac-specific genes and thereby regulates heart development. More recently, HOP/NECC1 was reported to be a suppressor of choriocarcinogenesis. Here, we examined the temporal expression profile of HOP/NECC1 in wild-type mouse placenta. We found that E8.5-E9.5 wild-type placenta expressed HOP/NECC1 in the giant cell and spongiotrophoblast layers. HOP/NECC1 (-/-) placenta exhibited marked propagation of giant cell layers and, in turn reduction of spongiotrophoblast formation. We demonstrated SRF transcriptional activity increased in the differentiating trophoblasts and forced expression of SRF in a trophoblast stem (TS) cell line induces the differentiation into giant cells. Negative regulation of SRF (serum response factor) by the binding of HOP/NECC1 protein contributed at least in part to the generation of these placental defects. Gradual induction of HOP/NECC1 in response to differentiation stimuli may result in the decision to differentiate into a particular type of trophoblastic cell lineage and result in non-lethal defects shown by the HOP/NECC1 (-/-) placentas.

Transcriptional Repressor Erf Determines Extraembryonic Ectoderm Differentiation

Extraembryonic ectoderm differentiation and chorioallantoic attachment are fibroblast growth factor (FGF)- and transforming growth factor beta-regulated processes that are the first steps in the development of the placenta labyrinth and the establishment of the fetal-maternal circulation in the developing embryo. Only a small number of genes have been demonstrated to be important in trophoblast stem cell differentiation. Erf is a ubiquitously expressed Erk-regulated, ets domain transcriptional repressor expressed throughout embryonic development and adulthood. However, in the developing placenta, after 7.5 days postcoitum (dpc) its expression is restricted to the extraembryonic ectoderm, and its expression is restricted after 9.5 dpc in a subpopulation of labyrinth cells. Homozygous deletion of Erf in mice leads to a block of chorionic cell differentiation before chorioallantoic attachment, resulting in a persisting chorion layer, a persisting ectoplacental cone cavity, failure of chorioallantoic attachment, and absence of labyrinth. These defects result in embryo death by 10.5 dpc. Trophoblast stem cell lines derived from Erf(dl1/dl1) knockout blastocysts exhibit delayed differentiation and decreased expression of spongiotrophoblast markers, consistent with the persisting chorion layer, the expanded giant cell layer, and the diminished spongiotrophoblast layer observed in vivo. Our data suggest that attenuation of FGF/Erk signaling and consecutive Erf nuclear localization and function is required for extraembryonic ectoderm differentiation, ectoplacental cone cavity closure, and chorioallantoic attachment.

New Light on Early Post-Implantation Pregnancy in the Mouse: Roles for Insulin-Like Growth Factor-II (IGF-II)?

Successful placental development and the associated changes to the decidual vasculature during early pregnancy are critical to pregnancy outcome. This study utilised immunohistochemistry to provide a photomicrographic account of trophoblast invasion, as well as the changes in the uterine vasculature in the decidua from days 5.5 to 10.5 of murine pregnancy. The pattern of trophoblast invasion during this time is particularly interesting because, unlike in humans, murine trophoblast giant cells (TGCs) do not invade the endometrium individually but remain in close contact with the expanding giant cell layer. Therefore, trophoblast cells are unlikely to play a direct role in remodelling the maternal vessels in early to mid pregnancy. Nevertheless, the decidual vessels appear to undergo extensive angiogenesis and remodelling to form a network of dilated vessels that presumably maximize placental blood supply. Importantly, the vessels closest to the conceptus lacked a smooth muscle layer throughout early pregnancy and therefore cannot strictly be described as spiral arterioles. TGCs may secrete molecules that can act to induce these vascular changes. Here we show that insulin-like growth factor-II (IGF-II) is expressed throughout early pregnancy in the entire conceptus including trophoblast cells, supporting its role in promoting early placental growth. In addition, the co-localisation of IGF-II and both IGF receptors in the developing blood vessels and/or adjacent stromal cells in the mesometrial, but not in the anti-mesometrial, decidua suggest that IGF-II, upon binding to one of these receptors, may play a role in both decidual angiogenesis and placental differentiation.

Differentiation of Trophoblast Giant Cells and Their Metabolic Functions Are Dependent on Peroxisome Proliferator-Activated Receptor β/δ

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

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.

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.

210. Localisation of insulin-like growth factor-II (IGF-II) and its receptor in early murine pregnancy: a role in placentation and angiogenesis in the decidua?

The highly invasive activity of the human placenta is tightly regulated by a variety of growth factors and other molecules. Contrary to the dominant view, recent data suggests that IGF-II, upon binding to the IGF2R, can stimulate an intracellular signalling pathway.1 Evidence in humans and mice suggests that IGF-II and the IGF2R are important regulators of placental growth; however, to date they have not yet been localised to early murine implantation sites. This study provides a photo micrographic account of early placental development and the decidual vasculature in the mouse, and localises IGF-II and the IGF2R from days 5.5 to 10.5 of pregnancy. During early pregnancy, the decidua displays a paucity of blood vessels, which appear to undergo angiogenesis, so that by day 10.5 the decidua has become a highly vascularized structure, with an extensive network of dilated vessels that presumably enable maximal blood supply to the placenta. Unlike humans, murine trophoblast cells do not invade the endometrium individually, but remain in close contact with the main giant cell layer. The trophoblast giant cells (TGCs) are the outermost cell type of the murine placenta and maternal blood spaces beneath this layer are not lined by endothelium. Due to their location, TGCs appear to play a direct role in displacing this endothelium and therefore may play a role in the transformation into trophoblast lined maternal blood spaces. IGF-II and its receptor were present throughout early pregnancy in the conceptus and maternal decidua supporting their role as regulators of fetal and placental development. Most interesting, however, was their association with the developing maternal blood vessels in the mesometrial decidua. It seems likely that in mice the maternal vessels are remodelled by a variety of locally derived molecules. By association, IGF-II and its receptor are likely candidates. (1)McKinnon T, et al. (2001). Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK. J. Clin. Endocrinol. Metab. 86(8), 3665–3674.

Keratin 8 protection of placental barrier function.

The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8−/− embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8−/− lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8−/− embryos. Finally, we show that K8−/− concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8−/− embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.

Tissue adhesion to bioactive glass-coated silicone tubing in a rat model of peritoneal dialysis catheters and catheter tunnels

Silicone peritoneal dialysis catheters do not develop tissue ingrowth, lack a mechanical barrier to periluminal bacterial migration and need cuffs for anchorage. We hypothesized that a bioactive glass coating composed of silicon, calcium, sodium and phosphorous oxides would cause a beneficial tissue reaction causing catheter adhesion, and tested this in a rat model. A hexane solvent-based method of coating silicone tubes with Bioglass powder was used, which maintained flexibility, and then the ultrastructure was confirmed with scanning electron microscopy (EM). Segments 2.5 cm were implanted subcutaneously in 8 Sprague-Dawley rats, with uncoated tubes as a contralateral control, and histology was done at 2, 4 and 6 weeks, including special stains and EM. The uncoated segments grossly had no adherence to surrounding tissue, and were physically separate from a thin fibrous capsule of approximately 50 micro width. Trichrome stains demonstrated the capsule was rich in collagen. There was minimal adjacent tissue reaction. In contrast, the coated tubes were palpably fixed to the soft tissues, and sections demonstrated an adjacent prominent layer of macrophages and multinucleated giant cells. Small numbers of lymphocytes were noted. This cellular reaction increased over the 6-week implant duration, and was also associated with neovascularization of the tissue adjacent to the segments (33 vessels in coated vs. 20 in controls per x 200 field, P < 0.0001). Many refractile silicone particles and prominent multinucleated giant cells were present, with small numbers of lymphocytes and macrophages. Stains showed scattered discontinuous calcific deposits. These findings are consistent with reports that the Bioglass(R) silicon oxide leads to the formation of a layer of hydroxyapatite, which binds to collagen and induces a tissue cellular reaction. In summary, bioactive glass coatings can improve the tissue retention of silicone tubing by promoting adhesion by collagen and cell proliferation, and are promising for future studies of peritoneal dialysis catheters.

Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development.

Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.

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 ∼150–300 μm 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.

Immunohistochemical distribution of epithelioid cell, myofibroblast, and transforming growth factor-beta1 in the granuloma caused by Mycobacterium avium intracellulare complex pulmonary infection.

The present study was designed to evaluate the distribution of epithelioid cells, myofibroblasts, and TGF-beta1 in the formation of granuloma caused by Mycobacterium avium intracellulare complex (MAC) lung infection. A retrospective study was performed for 9 cases of positive MAC culture in which lung resections were performed between January 1989 and August 1999. Resected lung specimens were evaluated histologically and immunohistochemically for CD68 (stain for monocytes and macrophages, and epithelioid cells) and alpha-smooth muscle actin as well as vimentin (stain for myofibroblasts), and TGF-beta1 was performed. When granuloma was initially formed, no myofibroblasts were found, but as caseous necrosis appeared, the thin epithelioid cell layer was detected and the outer myofibroblast layer gradually became thick. In the cavitary wall, the layer of epithelioid cells and multinucleated giant cells surrounded necrosis, and was associated with the outer layer of myofibroblasts. In addition, the anti-TGF-beta1 antibody stained the cytoplasm of epithelioid cells and multinucleated giant cells, preceding the advent of myofibroblasts. In summary, our present study evaluated distributions of epithelioid cells, myofibroblasts, and TGF-beta along with the morphogenesis of granuloma, and clearly demonstrated the immunohistochemical difference between granuloma with caseous necrosis and granulomas without caseous necrosis.

Pathological analysis of the cavitary wall in Mycobacterium avium intracellulare complex pulmonary infection.

The present study was designed to evaluate the process of cavity formation in Mycobacterium avium intracellulare complex (MAC) lung infection, pathologically and clinically. Using resected lung specimens, we first evaluated the distribution of MAC as well as the distribution of myofibroblasts in MAC lung infection according to several pathological findings classified as bronchiectasis, centrilobular nodules, cavity, nodules, bronchiolitis, or consolidation. Resected lung specimens (9 cases) were evaluated by special staining: Ziehl-Neelsen's method and immunohistochemically for CD68 (stain for monocytes and macrophages) and alpha-smooth muscle actin (stain for myofibroblasts). Chest CT findings were also examined in these 9 patients. In addition, the serial chest CT scans were reviewed in another 3 patients to evaluate the process of cavity formation, radiologically. Although extensive granuloma formations were observed in every pathological classification, MAC was demonstrated only in the necrotic tissue of the inner surface of the cavitary wall, which was connected to the airway. Myofibroblasts which expressed alpha-smooth muscle actin were intensely demonstrated in the cavitary wall compared with other pathological classifications. In the cavitary wall, the layer of epithelioid cells and multinucleated giant cells surrounded necrosis, and the layer of myofibroblasts surrounded the layer of epithelioid cells. Chest CT findings demonstrated that the cavitary walls were relatively thick. The evaluation of serial chest CT scans demonstrated that cavities were formed from previously existing nodules. Detection of mycobacteria in the cavitary wall, massive infiltration of myofibroblasts compared with other pathological classifications, and connection to the drainage bronchus, were believed to be important in the process of cavity formation in MAC pulmonary infection.

Srg3, a mouse homolog of yeast SWI3, is essential for early embryogenesis and involved in brain development.

Srg3 (SWI3-related gene product) is a mouse homolog of yeast SWI3, Drosophila melanogaster MOIRA (also named MOR/BAP155), and human BAF155 and is known as a core subunit of SWI/SNF complex. This complex is involved in the chromatin remodeling required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. We generated mice with a null mutation in the Srg3 locus to examine its function in vivo. Homozygous mutants develop in the early implantation stage but undergo rapid degeneration thereafter. An in vitro outgrowth study revealed that mutant blastocysts hatch, adhere, and form a layer of trophoblast giant cells, but the inner cell mass degenerates after prolonged culture. Interestingly, about 20% of heterozygous mutant embryos display defects in brain development with abnormal organization of the brain, a condition known as exencephaly. Histological examination suggests that exencephaly is caused by the failure in neural fold elevation, resulting in severe brain malformation. Our findings demonstrate that Srg3 is essential for early embryogenesis and plays an important role in the brain development of mice.

Nodal Regulates Trophoblast Differentiation and Placental Development

Nodal has been thought to be an embryo-specific factor that regulates development, but nodal is also expressed in the mouse placenta beginning at midgestation, specifically in the spongiotrophoblasts. In an insertional null nodal mutant, not only is embryonic development disrupted, but mouse placental development is also grossly altered with the loss of the diploid spongiotrophoblasts and labyrinth and an expansion of the polyploid giant cell layer. A hypomorphic mutation in nodal results in an expansion of the giant cell and spongiotrophoblast layers, and a decrease in labyrinthine development. Expression of nodal in trophoblast cell cultures is sufficient to inhibit trophoblast giant cell differentiation, demonstrating that nodal can act directly on trophoblasts. The mechanism of nodal action includes the inhibition of junB gene transcription. These results suggest that nodal may be involved in redirecting trophoblast fate towards the midgestational expansion of the labyrinth region while maintaining the thin layer of trophoblast giant cells and the underlying layer of spongiotrophoblasts that form the boundary between the maternal and extraembryonic compartments.

Placental failure in mice lacking the mammalian homolog of glial cells missing, GCMa.

The GCM family of transcription factors consists of Drosophila melanogaster GCM, an important regulator of gliogenesis in the fly, and its two mammalian homologs, GCMa and GCMb. To clarify the function of these mammalian homologs, we deleted GCMa in mice. Genetic ablation of murine GCMa (mGCMa) is embryonic lethal, with mice dying between 9.5 and 10 days postcoitum. At the time of death, no abnormalities were apparent in the embryo proper. Nervous system development, in particular, was not impaired, as might have been expected in analogy to Drosophila GCM. Instead, placental failure was the cause of death. In agreement with the selective expression of mGCMa in labyrinthine trophoblasts, mutant placentas did not develop a functional labyrinth layer, which is necessary for nutrient and gas exchange between maternal and fetal blood. Only a few fetal blood vessels entered the placenta, and these failed to thrive and branch normally. Labyrinthine trophoblasts did not differentiate. All other layers of the placenta, including spongiotrophoblast and giant cell layer, formed normally. Our results indicate that mGCMa plays a critical role in trophoblast differentiation and the signal transduction processes required for normal vascularization of the placenta.

Possible mechanism of congenital malformations induced by exposure of mouse preimplantation embryos to mitomycin C

ICR mice were treated intraperitoneally with mitomycin C at 5 mg/kg on day 3 of gestation. On day 18 of gestation, fetuses of treated dams were inspected for external, skeletal and visceral malformations. At 6 or 12 hr after mitomycin C treatment, the blastocysts were obtained from the uteri of treated dams and the degenerated cells within inner cell mass (ICM) and trophectoderm (TE) tissues were examined microscopically. On day 5, 8, 11, or 18 of gestation, the uteri of treated dams were obtained and those including embryos/fetuses and placentae were examined histologically. Finally, on each of gestational days 5-14, the blood of the treated dams was collected and the hematological parameters determined. Pre- and postimplantation losses in the dams treated with mitomycin C were significantly increased; increased frequency of abdominal wall defects and lumbar ribs in term fetuses, decreased fetal weight, and increased placental weight were noted as well. No significant increase in visceral malformations was found in term fetuses treated with mitomycin C. Frequency of degenerated cells within ICM and TE of blastocysts from dams treated with mitomycin C was significantly increased as compared with the controls. In dams treated with mitomycin C, decidua developed insufficiently and the trophoblast giant cell layer was not separated from the uterine lumen by maternal components; hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted. The number of erythrocytes, as well as hemoglobin concentration, hematocrit, and the percentage of reticulocytes in blood of dams treated with mitomycin C were significantly lower from days 6-12 of gestation, as compared with controls. The results of the present study showed that an increase in number of degenerated cells within blastocysts results in preimplantation loss and both maternal and embryonic hypoxia during major organogenesis results in postimplantation loss and congenital fetal malformations.

Identification of two new nonclassical members of the rat prolactin family.

The prolactin (PRL) family is comprised of a group of hormones/cytokines that are expressed in the anterior pituitary, uterus, and placenta. These proteins participate in the control of maternal and fetal adaptations to pregnancy. In this report, we have identified two new nonclassical members of the rat PRL family through a search of the National Center for Biotechnology Information dbEST database. The cDNAs were sequenced and their corresponding mRNAs characterized. Overall, the rat cDNAs showed considerable structural similarities with mouse proliferin-related protein (PLF-RP) and prolactin-like protein-F (PLP-F), consistent with their classification as rat homologs for PLF-RP and PLP-F. The expression of both cytokines/hormones was restricted to the placenta. The intraplacental sites of PLF-RP and PLP-F synthesis differed in the rat and the mouse. In the mouse, PLF-RP was expressed in the trophoblast giant cell layer of the midgestation chorioallantoic and choriovitelline placentas and, during later gestation, in the trophoblast giant cell and spongiotrophoblast layers within the junctional zone of the mouse chorioallantoic placenta. In contrast, in the rat, PLF-RP was first expressed in the primordium of the chorioallantoic placenta (ectoplacental cone region) and, later, exclusively within the labyrinth zone of the chorioallantoic placenta. In the mouse, PLP-F is an exclusive product of the spongiotrophoblast layer, whereas in the rat, trophoblast giant cells were found to be the major source of PLP-F, with a lesser contribution from spongiotrophoblast cells late in gestation. In summary, we have established the presence of PLF-RP and PLP-F in the rat.