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.     

Teratogenic effects of bis-diamine on early embryonic rat heart: an in vitro study

BACKGROUND: Bis-diamine induces cardiac defects, including conotruncal anomalies in rat embryos when the agent is administered to the mother. To evaluate the teratogenic effects and mechanism of bis-diamine, we performed morphological and immunohistochemical analyses of early rat embryos cultured in medium containing bis-diamine. METHODS: The embryos were removed from mother rats on gestational day 10.5 and cultured in medium containing 1 mg of bis-diamine for 6 hr. The embryos were then cultured in medium only for another 6, 12, 18, and 42 hr, corresponding to embryonic day (ED) 11.0, 11.25, 11.5, and 12.5, respectively. Some embryos from the same mothers were used as controls and were cultured in medium only for the corresponding periods to the embryos exposed to bis-diamine. Some mother rats were given a single oral dose of 200 mg of bis-diamine on gestational day 10.5. Embryos from these pregnant rats were removed 6 hr after the oral administration of bis-diamine, and were also cultured in medium only for 6, 12, 18, and 42 hr. RESULTS: No cardiac abnormalities were detected in the controls at any stage of development. Thirty-three of 51 (65%) embryos exposed to bis-diamine and 15 of 20 (75%) embryos removed from bis-diamine-administered mothers showed abnormal cardiac development, including dilated ventricle, elongation of outflow tract, and pericardial defect on ED 11.5. Four of six (67%) embryos exposed to bis-diamine, and five of seven (71%) removed from bis-diamine-administered mothers also presented almost the same cardiac abnormalities on ED 12.5. No cardiac abnormalities were detected in bis-diamine-treated embryos before ED 11.5. In addition, the expression of neural cell adhesion molecule (N-CAM) was examined using immunohistochemical methods. Fewer N-CAM immunoreactive cells were detected in the third and fourth aortic arches in the bis-diamine-treated embryos than in controls on ED 11.5. However, more N-CAM immunoreactive cells were detected in the bis-diamine-treated embryos than in controls on ED 12.5. CONCLUSIONS: These results suggest that bis-diamine induces cardiac anomalies by delaying the migration of neural crest cells into the heart and by disturbing the proliferation of pericardial precursor during early cardiac development.     

Expression of the acatalytic carbonic anhydrase VIII gene, Car8, during mouse embryonic development

Summary The carbonic anhydrase (CA)-like protein, CA VIII, lacks the typical carbon dioxide hydrase activity of the CA isozymes. However, the high degree of amino acid sequence similarity between the products of the mouse and the human CA VIII genes suggests an important biological function. We have attempted to investigate the function of this gene in mammalian development by conducting an in situ hybridization study on sagittal sections of mouse embryos at gestation days of 9.5–16.5 using a ³⁵S-labelled riboprobe. Results indicate that this gene (called Car8 in mice) is expressed as early as day 9.5 in a variety of organs including liver, branchial arches, neuroepithelium and developing myocardium. Between days 10.5 and 12.5, it showed a widespread distribution of mRNA expression that became more restricted as development progressed. The level of expression of Car8 mRNA was relatively high in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and the oronasal cavity. This revised version was published online in November 2006 with corrections to the Cover Date.     

Expression of fascin-1, an actin-bundling protein,in migrating hepatoblasts during rat liver development

Fascin-1 is an actin-bundling protein localized at the core actin bundles within microvillar projections and filopodial extensions in migrating cells. It is expressed at a low level in normal epithelial cells, but at a high level in tumor cells, indicating its importance in the invasion and motility of tumor cells. In addition, fascin-1 is expressed in human and murine embryos, occurring at high levels especially in developing nervous tissues. In this study, we have investigated the expression patterns of fascin-1 immunohistochemically during the early stages of rat hepatogenesis. A high expression of fascin-1 was detected in the liver bud and hepatoblasts at embryonic day (ED) 10.5, ED11.5, and ED12.5. Expression fell by ED13.5 and was not detectable at ED14.5. These observations demonstrate that the expression of fascin-1 is correlated with the migration activity of hepatoblasts during the early stages of liver development in rats. This study was supported in part by grant-in-aid for scientific research from the Japan Society for the Promotion of Science and by a grant from the Smoking Research Foundation, Japan.     

Teratogenic effects of bis-diamine on the developing cardiac conduction system

BACKGROUND: Congenital heart defects, including conotruncal anomalies, are often associated with arrhythmias. Bis-diamine induces conotruncal anomalies in embryos when administered to pregnant female rats. To investigate the mechanism of arrhythmia in conotruncal anomalies, we histologically examined the development of the cardiac conduction system in this animal model. METHODS: A single dose of 200 mg of bis-diamine was administered to pregnant Wistar rats on ED 10.5 of pregnancy. The embryos were removed on each day from ED 11.5 to 15.5. Immunoexpression of HNK-1, connexin40, and connexin43 were examined in serial sections. The distribution pattern of TUNEL-positive cells around the conduction system was also examined. RESULTS: HNK-1 immunoreactivity was evident in interventricular septum, in both the control and the bis-diamine-treated embryos from ED 12.5. Although a chain of connexin40-immunoreactive cells from interventricular septum to trabeculae, corresponding to the His bundle and its branches, was demonstrated at ED 13.5 in the control embryos, this chain was first detected at ED 14.5 in the bis-diamine-treated embryos. Immunoexpression of connexin43 in the working myocardium was also less in the bis-diamine-treated embryos than in the control at ED 13.5. The number of TUNEL-positive cells in the interventricular septum was highest at ED 12.5 in the control and at ED 13.5 in the bis-diamine-treated embryos. Furthermore, these TUNEL-positive cells were HNK-1 negative, vimentin-positive, and alpha smooth muscle actin-positive. CONCLUSIONS: Bis-diamine disturbed the normal development of gap junctions and apoptosis of myofibroblasts around the HNK-1-positive conduction tissue through overall poor myocardial proliferation and growth.     

In vivo hyperglycemia and its effect on Glut-1 expression in the embryonic heart

BACKGROUND: Maternal diabetes exposes embryos to periods of hyperglycemia. Glucose is important for normal cardiogenesis, and Glut-1 is the predominant glucose transporter in the embryo. METHODS: Pregnant mice were exposed to 6 or 12 hr hyperglycemia during organogenesis using intraperitoneal (IP) injections of D-glucose on gestational day (GD) 9.5 (plug = GD 0.5). Embryos were examined for morphology and total cardiac protein, and embryonic hearts were evaluated for Glut-1 protein and mRNA expression immediately after treatment (GD 9.75, GD 10.0), as well as on GD 10.5 and GD 12.5. RESULTS: IP glucose injections were effective in producing sustained maternal hyperglycemia. Maternal hyperglycemia for 6 or 12 hr on GD 9.5, followed by normoglycemia, produced a decrease in overall size and total cardiac protein in embryos evaluated on GD 10.5 but no difference on GD 12.5. Cardiac Glut-1 expression was immediately upregulated in embryos exposed to 6 or 12 hr maternal hyperglycemia. On GD 10.5, cardiac Glut-1 expression was not different in embryos exposed to maternal hyperglycemia for 6 hr but was downregulated in embryos exposed for 12 hr. On GD 12.5, cardiac Glut-1 expression in embryos exposed to maternal hyperglycemia on GD 9.5 for 6 or 12 hr, followed by normoglycemia, was not different from controls. The temporal pattern was the same for Glut-1 protein and mRNA expression. CONCLUSIONS: Hyperglycemia-induced alterations in Glut-1 expression likely interfere with balance of glucose available to the embryonic heart that may affect cardiac morphogenesis.     

Developmental expression of plasma glutathione peroxidase during mouse organogenesis

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.     

In vitro teratogenic potential of two antifungal triazoles: Triadimefon and triadimenol

Summary The teratogenic potential of two antifungal triazoles (Triadimefon and Triadimenol) has been investigated in vitro by the rat postimplantation whole embryo culture method. Rat embryos 9.5 d old were cultured for 48 h in rat serum with Triadimefon (12.5–250 μM) or Triadimenol (6.25–125 μM) and then examined. Some embryos exposed to Triadimenol (6.25–125 μM) were cultured for 12 extra hours in control serum to improve their developmental degree and then immunostain cranial nerves and ganglia. The exposure to the highest doses of triazoles only moderately reduced some morphometrical developmental parameters. By contrast, 25–250 μM Triadimefon and 25–125 μM Triadimenol induced specific concentration-related teratogenic effects at the level of first and second branchial arches. After immunostaining, embryos exposed to 12.5–125 μM Triadimenol showed specific cranial nerve and ganglia abnormalities. The possible implication of neural crest cell alterations on triazole-related abnormalities is discussed.     

Trophoblast glycogen cells differentiate early in the mouse ectoplacental cone: putative role during placentation

The role of differentiated trophoblast glycogen cells (GCs) in the ectoplacental cone (EPC) has not been elucidated yet. Recently, GC progenitors have been shown to be present from embryonic day 7.5 (E7.5), but glycogen is found in GC only from E10.5. Herein, we investigated the origin, localization and characterization of mouse GCs in EPC and their relationship with blood cells and trophoblast giant cells (TGCs) during placentation. Implantation sites (E5.5–E12.5) were processed for histological studies, histochemical detection (glycogen) and immunohistochemical staining (Ki67). Three-dimensional reconstruction of the EPC was obtained from suitably oriented embryos at E7.5. Our findings evidence that GCs are present and assembled in clusters from E6.5 to E12.5, and that they exhibit the classic vacuolated appearance and contain PAS-positive glycogen, which is amylase-sensitive and acetylation-resistant. In fact, only GCs were stained after acetylation, confirming unequivocally their presence in tissues. At E6.5, GCs showed numerous mitoses and vacuoles with scattered glycogen particles. At E7.5, GCs showed low numbers of mitoses and abundant vacuoles full of glycogen. During E7.5–E8.5, GCs were in close proximity to TGCs, and cells were intercalated by thin maternal blood spaces; placental GCs lost maternal blood contact during E9.5–E12.5. Our results indicate that GCs are originated and proliferate in the upper portion in the midregion of EPC at E6.5, and that at E7.5–E8.5 they show consistent glycogen deposits, which are likely metabolized to glucose. This compound may be directly transferred to circulating maternal blood, and used as a source of energy by GCs and TGCs during placentation.     

Hes1 regulates formations of the hypophyseal pars tuberalis and the hypothalamus

The hypophyseal pars tuberalis surrounds the median eminence and infundibular stalk of the hypothalamus as thin layers of cells. The pars tuberalis expresses MT1 melatonin receptor and participates in mediating the photoperiodic secretion of pituitary hormones. Both the rostral tip of Rathke’s pouch (pars tuberalis primordium) and the pars tuberalis expressed αGSU mRNA, and were immunoreactive for LH, chromogranin A, and TSHβ in mice. Hes genes control progenitor cell differentiation in many embryonic tissues and play a crucial role for neurulation in the central nervous system. We investigated the Hes1 function in outgrowth and differentiation of the pars tuberalis by using the markers for the pars tuberalis. In homozygous Hes1 null mutant embryos, the rostral tip was formed in the basal-ventral part of Rathke’s pouch at embryonic day (E)11.5 as well as in wild-type embryos. In contrast to the wild-type, the rostral tip of null mutants could not extend rostrally with age; it remained in the low extremity of Rathke’s pouch during E12.5–E13.5 and disappeared at E14.5, resulting in lack of the pars tuberalis. Development of the ventral diencephalon was impaired in the null mutants at early stages. Rathke’s pouch, therefore, could not link with the nervous tissue and failed to receive inductive signals from the diencephalon. In a very few mutant mice in which the ventral diencephalon was partially sustained, some pars tuberalis cells were distributed around the hypoplastic infundibulum. Thus, Hes1 is required for development of the pars tuberalis and its growth is dependent on the ventral diencephalon.     

Ultrasound diagnosis of mouse pregnancy and gestational staging

Phenotypic analysis of mutant mice is limited by lack of accurate, simple, and nondestructive in utero imaging techniques. This study evaluated the usefulness of ultrasound imaging (US) to stage fetal mouse gestational age (GA) and depict morphologic development. We imaged 16 pregnant CD-1 mice and a total of 92 fetuses with a 15-MHz US transducer from 9.5 d postcoitus (DPC) until 20.5 DPC or delivery. Parameters recorded included gestational sac dimensions, crown-rump length (CRL), biparietal diameter (BPD), thoracoabdominal diameter (TAD), onset of cardiac activity, and morphologic development. At 9.5 d DPC, all gestations appeared as rounded sacs, with a diameter (mean +/- standard error) of 4.4 +/- 1 mm. BPD, CRL, and GA were highly correlated. The following structures were first identifiable at the following GA: cardiac activity, 10.5 DPC; major cardiovascular structures, 11.5 DPC; limb buds, 10.5 DPC; spine, 12.5 DPC; face and skull ossification, 13.5 DPC; rib ossification, 15.5 DPC; hind- and forelimb digits, 15.5 DPC; stomach and urinary bladder, 17.5 DPC; visualization of the rhombencephalic vesicle, 13.5 DPC; and visualization of the lateral ventricles, 14.5 DPC. The echogenic lungs were distinct from the liver as early as 12.5 DPC. The circle of Willis was detectable with color Doppler as early as 13.5 DPC and was easily visualized at 15.5 DPC. We found that US provides accurate, simple staging criteria for fetal mouse gestational development after 9.5 DPC and may be a nondestructive means of documenting phenotypic alterations in mutant mice in utero.     

Localization and subcellular distribution of prolyl oligopeptidase in the mouse placenta

Prolyl oligopeptidase (POP) is a serine endopeptidase which selectively digests a -Pro-X- peptide bond. Our previous study showed that POP mRNA was strongly expressed in the spongiotrophoblast of the mouse placenta at E17.5, suggesting its importance in development. To gain more insight into POP’s role during gestation, we investigated its expression using different developmental stages of placenta. As a result of in situ hybridization, we found that localization of POP mRNA changed at E12.5. POP mRNA was strongly expressed in the spongiotrophoblast and labyrinth at E10.5 and E11.5 but thereafter only in the spongiotrophoblast. Immunohistochemistry revealed that POP was present in the parietal trophoblast giant cell, the spongiotrophoblast cell, and the labyrinth at E11.5 but the strong expression in the labyrinth was maintained only in the canal-associated and sinusoidal trophoblast giant cells at E16.5 and E18.5. To determine subcellular distribution of the POP protein, we fractionated the placental extract into cytoplasmic, membrane, and nuclear subfractions. By Western blot analysis, POP was detected in the cytoplasmic and membrane fractions but not in the nuclear fraction at E11.5 and E16.5. Interestingly, the cytoplasmic POP exhibited higher enzymatic activity than the membrane-associated type. These data suggest that the cytoplasmic and membrane-associated POP have distinct roles in different types of placental cells.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.