Dynamic expression of manganese superoxide dismutase during mouse embryonic organogenesis

The balance between reactive oxygen species production and antioxidant defense enzymes in embryos is necessary for normal embryogenesis. To determine the dynamic expression profile of manganese superoxide dismutase (MnSOD) in embryos, which is an essential antioxidant enzyme in embryonic organogenesis, the expression level and distribution of MnSOD mRNA and protein were investigated in mouse embryos, as well as extraembryonic tissues on embryonic days (EDs) 7.5-18.5. MnSOD mRNA levels were remarkably high in extraembryonic tissues rather than in embryos during these periods. MnSOD protein levels were also higher in extraembryonic tissues than in embryos until ED 16.5, but the opposite trend was found after ED 17.5. MnSOD mRNA was observed in the chorion, allantois, amnion, ectoderm, ectoplacental cone and neural fold at ED 7.5 and in the neural fold, gut, ectoplacental cone, outer extraembryonic membranes and primitive heart at ED 8.5. After removing the extraembryonic tissues, the prominent expression of MnSOD mRNA in embryos was seen in the sensory organs, central nervous system and limbs on EDs 9.5-12.5 and in the ganglia, spinal cord, sensory organ epithelia, lung, blood cells and vessels, intestinal and skin epithelia, hepatocytes and thymus on EDs 13.5-18.5. Strong MnSOD immunoreactivity was observed in the choroid plexus, ganglia, myocardium, blood vessels, heapatocytes, pancreatic acinus, osteogenic tissues, brown adipose tissue, thymus and skin. These findings suggest that MnSOD is mainly produced from extraembryonic tissues and then may be utilized to protect the embryos against endogenous or exogenous oxidative stress during embryogenesis.     

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.     

Spatiotemporal expression of the selenoprotein P gene in postimplantational mouse embryos

Selenoprotein P (Sepp) is an extracellular glycoprotein which functions principally as a selenium (Se) transporter and antioxidant. In order to assess the spatiotemporal expression of the Sepp gene during mouse embryogenesis, quantitative RT-PCR and in situ hybridization analyses were conducted in embryos and extraembryonic tissues, including placenta. Sepp mRNA expression was detected in all embryos and extraembryonic tissues on embryonic days (E) 7.5 to 18.5. Sepp mRNA levels were high in extraembryonic tissues, as compared to embryos, on E 7.5-13.5. However, the levels were higher in embryos than in extraembryonic tissues on E 14.5-15.5, but were similar in both tissues during the subsequent periods prior to birth. According to the results of in situ hybridization, Sepp mRNA was expressed principally in the ectoplacental cone and neural ectoderm, including the neural tubes and neural folds. In whole embryos, Sepp mRNA was expressed abundantly in nervous tissues on E 9.5-12.5. Sepp mRNA was also expressed in forelimb and hindlimb buds on E 10.5-12.5. In the sectioned embryos, on E 13.5-18.5, Sepp mRNA was expressed persistently in the developing limbs, gastrointestinal tract, nervous tissue, lung, kidney and liver. On E 16.5-18.5, Sepp mRNA expression in the submandibular gland, whisker follicles, pancreas, urinary bladder and skin was apparent. In particular, Sepp mRNA was detected abundantly in blood cells during all the observed developmental periods. These results show that Sepp may function as a transporter of selenium, as well as an antioxidant, during embryogenesis.     

Tissue-restricted expression of thrombomodulin in the placenta rescues thrombomodulin-deficient mice from early lethality and reveals a secondary developmental block

The endothelial cell surface receptor thrombomodulin (TM) inhibits blood coagulation by forming a complex with thrombin, which then converts protein C into the natural anticoagulant, activated protein C. In mice, a loss of TM function causes embryonic lethality at day 8.5 p.c. (post coitum) before establishment of a functional cardiovascular system. At this developmental stage, TM is expressed in the developing vasculature of the embryo proper, as well as in non-endothelial cells of the early placenta, giant trophoblast and parietal endoderm. Here, we show that reconstitution of TM expression in extraembryonic tissue by aggregation of tetraploid wild-type embryos with TM-null embryonic stem cells rescues TM-null embryos from early lethality. TM-null tetraploid embryos develop normally during midgestation, but encounter a secondary developmental block between days 12.5 and 16.5 p.c. Embryos lacking TM develop lethal consumptive coagulopathy during this period, and no live embryos are retrieved at term. Morphogenesis of embryonic blood vessels and other organs appears normal before E15. These findings demonstrate a dual role of TM in development, and that a loss of TM function disrupts mouse embryogenesis at two different stages. These two functions of TM are exerted in two distinct tissues: expression of TM in non-endothelial extraembryonic tissues is required for proper function of the early placenta, while the absence of TM from embryonic blood vessel endothelium causes lethal consumptive coagulopathy.     

Defective brain development in mice lacking the Hif-1alpha gene in neural cells

Hypoxia-inducible factor 1alpha (HIF-1alpha) is essential for vascular development during embryogenesis and pathogenesis. However, little is known about its role in brain development. To investigate the function of HIF-1alpha in the central nervous system, a conditional knockout mouse was made with the Cre/LoxP system with a nestin promoter-driven Cre. Neural cell-specific HIF-1alpha-deficient mice exhibit hydrocephalus accompanied by a reduction in neural cells and an impairment of spatial memory. Apoptosis of neural cells coincided with vascular regression in the telencephalon of mutant embryos, and these embryonic defects were successfully restored by in vivo gene delivery of HIF-1alpha to the embryos. These results showed that expression of HIF-1alpha in neural cells was essential for normal development of the brain and established a mouse model that would be useful for the evaluation of therapeutic strategies for ischemia, including hypoxia-mediated hydrocephalus.     

小鼠早期胚胎发育期间LIF基因表达的研究(简报)

白血病抑制因子(Leukemia inhibitory fac-tor,LIF)是近年来研究较为广泛的细胞生长调节因子之一。最初发现LIF能够在体外诱导小鼠髓样白血病细胞株M1细胞向正常细胞分化,进一步分离纯化蛋白以及克隆基因后发现LIF在体外还具有多种功能,作用于不同的靶细胞时引起的生理效应也各不相同。目前已知的功能有:刺激肝脏细胞急性期反应蛋白的     

Activity-regulated, cytoskeleton-associated protein (Arc) is essential for visceral endoderm organization during early embryogenesis

Activity-regulated, cytoskeleton-associated protein (Arc) was first identified as an immediate-early gene regulated by synaptic activity. We have studied its functional role in vivo using a gene-targeting approach. We found that Arc is encoded by a single exon, and Arc mRNA is ubiquitously expressed in early mouse embryos. Homozygous Arc mutants are severely growth-retarded, fail to gastrulate and subsequently die before day 8.5 of embryogenesis. Further analysis revealed severe disorganization of visceral endoderm formation, and total separation and ectopic location of embryonic and extraembryonic structure. These findings demonstrate that Arc function is essential for early embryo development and patterning in mice, and support the hypothesis that signaling from visceral endoderm is essential for normal patterning of the extraembryonic and embryonic structure.     

Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population

During embryogenesis the central and peripheral nervous systems arise from a neural precursor population, neurectoderm, formed during gastrulation. We demonstrate the differentiation of mouse embryonic stem cells to neurectoderm in culture, in a manner which recapitulates embryogenesis, with the sequential and homogeneous formation of primitive ectoderm, neural plate and neural tube. Formation of neurectoderm occurs in the absence of extraembryonic endoderm or mesoderm and results in a stratified epithelium of cells with morphology, gene expression and differentiation potential consistent with positionally unspecified neural tube. Differentiation of this population to homogeneous populations of neural crest or glia was also achieved. Neurectoderm formation in culture allows elucidation of signals involved in neural specification and generation of implantable cell populations for therapeutic use.     

The development potential of parthenogenetically derived cells in chimeric mouse embryos: implications for action of imprinted genes

Parthenogenetic embryos of mice die shortly after implantation and characteristically contain poorly developed extraembryonic tissue. To investigate the basis of the abnormal development of parthenotes, we combined them with normal embryos to produce chimeras and examined the distribution of the parthenogenetically derived cells during preimplantation and early postimplantation development. The parthenogenetic embryos were derived from a transgenic mouse line bearing a large insert, which allowed these cells to be identified in histological sections using in situ hybridization. At the blastocyst stage, the parthenogenetic embryos contributed cells to the trophectoderm (TE) and inner cell mass (ICM) of chimeras. By 6.5 days, however, in almost every embryo, parthenogenetically derived cells were not detected in the extraembryonic trophoblast tissue descended from the TE. In contrast, parthenogenetically derived cells could contribute to all descendants of the ICM of 6.5-and 7.5-day chimeras, including the extraembryonic visceral and parietal endoderm. Quantitative analysis of the degree of chimerism in the embryonic ectoderm at 6.5-7.5 days indicated that parthenogenetically derived cells could contribute as extensively as normal cells. These results indicate that normal trophoblast development requires gene expression from the paternally inherited genome before 6.5 days of embryogenesis. Tissues of the ICM lineage, however, apparently can develop independently of the paternal genome at least to 7.5 days of embryogenesis. Comparison of these results with those of others suggests that the influence of imprinted genes is manifested at different times and in a variety of tissues during development.     

Expression of HSG is essential for mouse blastocyst formation

It has been shown recently that hyperplasia suppressor gene (HSG) is a powerful regulator for cell proliferation and has a critical role in mitochondrial fusion in many cells. However, little is known about its expression, localization, and function during oocyte maturation and early embryogenesis. In this study, with indirect immunofluorescent staining and Western blotting, we found that HSG was expressed in mouse oocytes and preimplantation embryos which primarily exhibited a submembrane distribution pattern in the cytoplasm. Moreover, HSG mainly associated with beta-tubulin during oocyte maturation and early embryonic development. When mouse zygotes were injected with HSG antisense plasmid and cultured in vitro, their capacity to form blastocysts was severely impaired. Our results indicate that HSG plays an essential role in mouse preimplantation development.     

Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse.

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, we have generated chimeric mouse embryos and performed tissue layer recombination explant assays. In chimeric embryos in which the visceral endoderm was composed of predominantly wild-type cells, we found that Lim1(-)(/)(-) cells were able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but that embryonic day 9.5 chimeric embryos displayed a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1(-)(/)(-) embryonic stem cells into wild-type tetraploid blastocysts lacked forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1(-)(/)(-) embryos was unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm was composed of predominantly Lim1(-)(/)(-) cells and the embryo proper of largely wild-type cells, also phenocopied the Lim1(-)(/)(-) headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. We discuss a double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse.     

BMP signaling through ACVRI is required for left-right patterning in the early mouse embryo

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse.     

Somatic Donor Cell Type Correlates with Embryonic,but Not Extra-Embryonic,Gene Expression in Postimplantation Cloned Embryos

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10^–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.     

Differential dysmorphogenesis induced by microinjection of an alkylating agent into rat conceptuses cultured in vitro

A technique of microinjection of small quantities of teratogens into extraembryonic compartments or specific organ primordium of rat conceptuses of pregnancy day 11 is described. Conceptuses microinjected with 50 nl tissue culture medium developed normally for 44-45 hr when cultured in homologous rat serum, indicating that the microinjection procedure itself did not produce any deleterious effects on growth and differentiation of embryos. Microinjection of an alkylating agent, phosphoramide mustard dissolved in tissue culture medium, into the exocoelom produced anomalous embryogenesis, consisting of retarded embryonic growth, anomalies of the neural tube, and general necrosis of various organ primordia. In contrast, the embryonic development remained relatively unaffected by microinjection of identical amounts of this alkylating agent into the amniotic cavity. However, neural-tube differentiation was markedly affected when phosphoramide mustard was injected into anterior neural-tube fluid, producing anencephalic or microcephalic embryos without significant effect on postcephalic organ differentiation. The morphogenesis of the anterior limb was unaffected by local injection of the agent into somitic tissues adjacent to the presumptive limb-bud region. Therefore, it appears that differential dysmorphogenesis could be induced by microinjection of an alkylating agent into different conceptus compartments. These results indicate that even during early embryogenesis various cell types are not equally susceptible to a given teratogen, and that the differential cytotoxicity of the teratogen toward specific embryonic or extraembryonic cells and tissues may account for embryonic anomalies characteristically produced by that agent.