CD-1 Mouse Embryos Research Articles

Establishing protein expression profiles involved in tooth development using a proteomic approach.

Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.

Effects of Insulin-Like Growth Factor 1 on the In Vitro Development of Mouse Embryos after Cryopreservation

Cryopreservation of gametes and embryos is widely used for mammalian genome resource banking. Effects of insulin-like growth factor-1 (IGF-1) on the in vitro development of cleavage stage CD1 mouse embryos after their cryopreservation were studied. The mouse embryos developed in vivo up to the 4–6-cell stage were frozen in a programmable freezer according to standard protocol with 10% propylene glycol as a cryoprotectant and thereafter were thawed and cultured in vitro in KSOM for 48 h. The percentage of embryos developed up to blastocyst stage in culture medium supplemented with IGF-1 (40 ng/mL) was higher as compared to controls (78.9 and 47.4%, respectively, p < 0.01). Moreover, blastocysts developed in the presence of IGF-1 contained more cells as compared to controls (79.8 ± 3.9 and 58.6 ± 3.6 cells, respectively, p < 0.001). However, the index of nuclear fragmentation did not differ between these groups. Experimental results presented here revealed the stimulating effects of IGF-1 on the mouse preimplantation embryos after their cryopreservation.

Effects of an Environmentally Relevant Mixture of Organophosphate Esters Derived From House Dust on Endochondral Ossification in Murine Limb Bud Cultures.

Organophosphate esters (OPEs) are used widely as flame retardants and plasticizers but much remains unknown about their potential toxicity. Previously, we reported that 4 individual OPEs suppress endochondral ossification in murine limb bud cultures. However, real-life exposure is to complex OPE mixtures. In the present study, we tested the hypothesis that a Canadian household dust-based OPE mixture will affect endochondral ossification in gestation day 13 CD1 mouse embryo limb buds expressing fluorescent markers for the major cell populations involved in the process: collagen type II alpha 1-enhanced cyan fluorescent protein (proliferative chondrocytes), collagen type X alpha 1-mCherry (hypertrophic chondrocytes), and collagen type I alpha 1-yellow fluorescent protein (osteoblasts). Limbs were cultured for 6 days in the presence of vehicle or dilutions of the OPE mixture (1/1 000 000, 1/600 000, and 1/300 000). All 3 OPE mixture dilutions affected cartilage template development and the progression of endochondral ossification, as indicated by the fluorescent markers. The expression of Sox9, the master regulator of chondrogenesis, was unchanged, but the expression of Runx2 and Sp7, which drive chondrocyte hypertrophy and osteoblastogenesis, was dilution-dependently suppressed. RNA-seq revealed that exposure to the 1/300 000 dilution of the OPE mixture for 24 h downregulated 153 transcripts and upregulated 48 others by at least 1.5-fold. Downregulated transcripts were enriched for those related to the immune system and bone formation. In contrast, upregulated transcripts were enriched for those with stress response functions known to be regulated by ATF4 activation. Thus, exposure to the mixture of OPEs commonly found in house dust may have adverse effects on bone formation.

Characterizing the effects of in utero valproic acid exposure on NF-κB signaling in CD-1 mouse embryos during neural tube closure

Nuclear factor kappa B (NF-κB) is a heterodimer of protein subunits p65 and p50, that regulates the expression of a large number of genes related to cell growth and proliferation. The p65 subunit is activated after phosphorylation by Pim-1, while the p50 subunit is the cleaved product of its precursor molecule p105. Valproic acid (VPA), an antiepileptic drug, is a known teratogen and its exposure during pregnancy is associated with 1–2% of neural tube defects in the offspring. The current study aimed at investigating the effects of in utero VPA exposure on the key components of the NF-κB signaling pathway including p65, p50, and Pim-1 in CD-1 mouse embryos during the critical period of neural tube closure. Here we report that p65, Pim-1 and p105/p50 mRNA were significantly (p < 0.05) downregulated at 1 and 3 h following in utero exposure to a teratogenic dose (400 mg/kg) of VPA in gestational day (GD)9 exposed embryos. At GD13 heads of control, non-exencephalic and exencephalic embryos were used for analysis and we found significant upregulation of p65 protein expression in non-exencephalic GD13 heads while p50 protein levels were significantly downregulated in both non-exencephalic and exencephalic groups. On the other hand, p65 and p50 protein levels remained unchanged in the nuclear extracts of the VPA-exposed non-exencephalic and exencephalic GD13 embryo heads. The reported results suggest that VPA exposure perturbates p65, p105/p50, Pim-1 transcript and p65/p50 protein levels in mouse embryos.

P.112 Effects of early-life fluvoxamine exposure on social behaviours of white rats depend on the timing of its perinatal administration

Valproic acid (VPA), an antiepileptic and mood-stabilizing drug, is prescribed to women of reproductive age. VPA is associated with a 1–2% increase in neural tube defects in offspring following gestational exposure and results in epigenetic modifications induced by perturbations in transcription cofactors. Cbp and p300, two transcription cofactors, play key roles in embryonic neural development. p300 is a downstream target of Akt, a protein kinase B associated with cell survival and anti-apoptotic mechanisms, as part of the Akt-p300 axis. We examined the effects of in utero VPA exposure on Cbp, p300, and Akt in gestational day (GD)9, GD10 and GD13 CD-1 mouse embryos following a teratogenic maternal dose of 400 mg/kg. Embryos were collected at 0, 1, 3 and 6 h post-dosing on GD9, 24 h post-dosing on GD10 and on GD13. GD10 embryos were grouped according to the status of neural tube closure in control, closed and open groups. GD13 heads were grouped as control, exposed but non-exencephalic and exencephalic. Our data indicate that Cbp, p300 and Akt mRNA levels were downregulated at 1 and 3 h post-exposure in GD9 embryos while Cbp and p300 protein levels remained stable. Akt protein levels were significantly increased 1 h post-exposure. No significant changes were observed in either mRNA or protein expression in embryos with closed or open neural tubes compared to the control group at GD10. Downregulated expression of Cbp, p300, and Akt may play a key role in VPA-induced neural tube defects considering their vitally important role in embryonic development.

Exposure to tert-Butylphenyl Diphenyl Phosphate, an Organophosphate Ester Flame Retardant and Plasticizer, Alters Hedgehog Signaling in Murine Limb Bud Cultures.

Organophosphate esters have become widely used as flame retardants since the phase out of polybrominated diphenyl ethers. Previously, we demonstrated that some organophosphate esters, such as tert-butylphenyl diphenyl phosphate (BPDP), were more detrimental to endochondral ossification in murine limb bud cultures than one of the major polybrominated diphenyl ethers that they replaced, 2,2',4,4'-tetrabromodiphenyl ether. Here, we used a transcriptomic approach to elucidate the mechanism of action of BPDP in the developing limb. Limb buds collected from gestation day 13 CD1 mouse embryos were cultured for 3 or 24 h in the presence of vehicle, 1 μM, or 10 μM BPDP. RNA sequencing analyses revealed that exposure to 1 µM BPDP for 24 h increased the expression of 5 transcripts, including Ihh, and decreased 14 others, including Gli1, Ptch1, Ptch2, and other targets of Hedgehog (Hh) signaling. Pathway analysis predicted the inhibition of Hh signaling. Attenuation of Hh signaling activity began earlier and reached a greater magnitude after exposure to 10 µM BPDP. Because this pathway is part of the regulatory network governing endochondral ossification, we used a known Hh agonist, purmorphamine, to determine the contribution of Hh signaling inhibition to the negative impact of BPDP on endochondral ossification. Cotreatment of limbs with purmorphamine rescued the detrimental morphological changes in the cartilage template induced by BPDP exposure though it did not restore the expression of key transcription factors, Runx2 and Sp7, to control levels. These data highlight Hh signaling as a developmentally important pathway vulnerable to environmental chemical exposures.

Role of Cbp, p300 and Akt in valproic acid induced neural tube defects in CD-1 mouse embryos

Valproic acid (VPA), an antiepileptic and mood-stabilizing drug, is prescribed to women of reproductive age. VPA is associated with a 1–2% increase in neural tube defects in offspring following gestational exposure and results in epigenetic modifications induced by perturbations in transcription cofactors. Cbp and p300, two transcription cofactors, play key roles in embryonic neural development. p300 is a downstream target of Akt, a protein kinase B associated with cell survival and anti-apoptotic mechanisms, as part of the Akt-p300 axis. We examined the effects of in utero VPA exposure on Cbp, p300, and Akt in gestational day (GD)9, GD10 and GD13 CD-1 mouse embryos following a teratogenic maternal dose of 400 mg/kg. Embryos were collected at 0, 1, 3 and 6 h post-dosing on GD9, 24 h post-dosing on GD10 and on GD13. GD10 embryos were grouped according to the status of neural tube closure in control, closed and open groups. GD13 heads were grouped as control, exposed but non-exencephalic and exencephalic. Our data indicate that Cbp, p300 and Akt mRNA levels were downregulated at 1 and 3 h post-exposure in GD9 embryos while Cbp and p300 protein levels remained stable. Akt protein levels were significantly increased 1 h post-exposure. No significant changes were observed in either mRNA or protein expression in embryos with closed or open neural tubes compared to the control group at GD10. Downregulated expression of Cbp, p300, and Akt may play a key role in VPA-induced neural tube defects considering their vitally important role in embryonic development.

Gestational exposure to valproic acid upregulates total Stat3 protein expression while downregulating phosphorylated Stat3 in CD-1 mouse embryos with neural tube defects.

Valproic acid (VPA), a widely prescribed antiepileptic drug and an effective treatment for psychiatric disorders, is teratogenic causing neural tube defects (NTDs) and other defects in the exposed embryo. Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is activated via tyrosine phosphorylation. Stat3, as well as its active form (pYStat3), is expressed during neural tube closure in murine development. This study investigated the effects of in utero VPA exposure on embryonic Stat3 mRNA and protein expression during the critical period of neural tube closure in CD-1 mouse embryos. Following the exposure of CD-1 pregnant mice to the teratogenic dose of 400 mg/kg VPA or saline on gestational day (GD) 9, embryos were harvested at 1, 3, 6, or 24 hr and on GD13. Stat3 mRNA levels remained unchanged at all time points. Total Stat3 protein levels were significantly (p < .05) increased in GD9 embryos at 1 and 6 hr post-exposure and in GD13 exposed nonexencephalic and exencephalic embryo heads. In contrast, phosphorylated Stat3 levels were significantly (p < .05) downregulated in GD9 embryos at the 3 and 6 hr time points with an overall trend of downregulation in the GD10 and GD13 groups. Total and phosphorylated Stat3 protein levels remained unchanged in nuclear extracts of the exposed nonexencephalic and exencephalic GD13 embryo heads. The reported significant downregulation of phosphorylated Stat3 levels suggests its possible role in VPA-induced NTDs in mouse embryos.

Valproic acid increases NF-κB transcriptional activation despite decreasing DNA binding ability in P19 cells, which may play a role in VPA-initiated teratogenesis

The nuclear factor-kappa B (NF-κB) family of transcription factors regulate gene expression in response to diverse stimuli. We previously demonstrated that valproic acid (VPA) exposure in utero decreases total cellular protein expression of the NF-κB subunit p65 in CD-1 mouse embryos with a neural tube defect but not in phenotypically normal littermates. This study evaluated p65 mRNA and protein expression in P19 cells and determined the impact on DNA binding ability and activity. Exposure to 5mM VPA decreased p65 mRNA and total cellular protein expression however, nuclear p65 protein expression was unchanged. VPA reduced NF-κB DNA binding and nuclear protein of the p65 DNA-binding partner, p50. NF-κB transcriptional activity was increased with VPA alone, despite decreased phosphorylation of p65 at Ser276, and when combined with tissue necrosis factor α. These results demonstrate that VPA increases NF-κB transcriptional activity despite decreasing DNA binding, which may play a role in VPA-initiated teratogenesis.

Enhanced NADPH oxidases and reactive oxygen species in the mechanism of methanol-initiated protein oxidation and embryopathies in vivo and in embryo culture.

Methanol (MeOH) teratogenicity in rodents may be mediated in part by reactive oxygen species (ROS), the source of which is unknown. To determine if MeOH enhances embryonic ROS-producing NADPH oxidases (NOXs), p22phox mRNA and protein and oxidatively damaged protein were measured in gestational day 12 MeOH-exposed CD-1 mouse embryos with or without pretreatment with the free radical spin trap phenylbutylnitrone (PBN) or the NOX inhibitor diphenyleneiodonium chloride (DPI). MeOH exposure upregulated p22phox mRNA and protein expression, and enhanced protein oxidation, within 3-6 h. Compared to embryos exposed to MeOH alone, PBN and DPI pretreatment decreased MeOH-enhanced p22phox mRNA expression, DPI but not PBN blocked p22phox protein expression, and both blocked protein oxidation. To assess developmental relevance, mouse embryos were exposed in culture for 24 h to MeOH or vehicle with or without pretreatment with PBN, DPI, or the prostaglandin H synthase (PHS) inhibitor eicosatetraynoic acid (ETYA), and evaluated for abnormalities. ETYA did not prevent MeOH embryopathies, despite blocking phenytoin embryopathies (ROS-initiating positive control), precluding bioactivation of MeOH or its metabolites by PHS. Concentration-dependent MeOH embryopathies were blocked by both DPI and PBN pretreatment, suggesting that enhanced embryonic NOX-catalyzed ROS formation and oxidative stress may contribute to the mechanism of MeOH embryopathies.

Inhibition of fumonisin B1 cytotoxicity by nanosilicate platelets during mouse embryo development.

Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.

High Frequency Ultrasound for In Vivo Pregnancy Diagnosis and Staging of Placental and Fetal Development in Mice

BackgroundUltrasound is a valuable non-invasive tool used in obstetrics and gynecology to monitor the growth and well being of the human fetus. The laboratory mouse has recently emerged as an appropriate model for fetal and perinatal studies because morphogenetic processes in mice exhibit adequate homology to those in humans, and genetic manipulations are relatively simple to perform in mice. High-frequency ultrasound (HFUS) has recently become available for small animal preclinical imaging and can be used to study pregnancy and development in the mouse. The objective of the current study was to assess the main applications of HFUS in the evaluation of fetal growth and placental function and to better understand human congenital diseases.Methodology/Principal FindingsOn each gestational day, at least 5 dams were monitored with HFUS; a total of ∼200 embryos were examined. Because it is not possible to measure each variable for the entire duration of the pregnancy, the parameters were divided into three groups as a function of the time at which they were measured. Univariate analysis of the relationship between each measurement and the embryonic day was performed using Spearman’s rank correlation (Rs). Continuous linear regression was adopted for multivariate analysis of significant parameters. All statistical tests were two-sided, and a p value of 0.05 was considered statistically significant.Conclusions/SignificanceThe study describes the main applications of HFUS to assess changes in phenotypic parameters in the developing CD1 mouse embryo and fetus during pregnancy and to evaluating physiological fetal and placental growth and the development of principal organs such as the heart, kidney, liver, brain and eyes in the embryonic mouse. A database of normal structural and functional parameters of mouse development will provide a useful tool for the better understanding of morphogenetic and cardiovascular anomalies in transgenic and mutant mouse models.

Effects of a ketogenic diet during pregnancy on embryonic growth in the mouse

BackgroundThe increasing use of the ketogenic diet (KD), particularly by women of child-bearing age, raises a question about its suitability during gestation. To date, no studies have thoroughly investigated the direct implications of a gestational ketogenic diet on embryonic development.MethodsTo fill this knowledge gap we imaged CD-1 mouse embryos whose mothers were fed either a Standard Diet (SD) or a KD 30 days prior to, as well as during gestation. Images were collected at embryonic days (E) 13.5 using Optical Projection Tomography (OPT) and at E17.5 using Magnetic Resonance Imaging (MRI).ResultsAn anatomical comparison of the SD and KD embryos revealed that at E13.5 the average KD embryo was volumetrically larger, possessed a relatively larger heart but smaller brain, and had a smaller pharynx, cervical spinal cord, hypothalamus, midbrain, and pons, compared with the average SD embryo. At E17.5 the KD embryo was found to be volumetrically smaller with a relatively smaller heart and thymus, but with enlarged cervical spine, thalamus, midbrain and pons.ConclusionA ketogenic diet during gestation results in alterations in embryonic organ growth. Such alterations may be associated with organ dysfunction and potentially behavioral changes in postnatal life.

Toward Routine Use of 3D Histopathology as a Research Tool

Three-dimensional (3D) reconstruction and examination of tissue at microscopic resolution have significant potential to enhance the study of both normal and disease processes, particularly those involving structural changes or those in which the spatial relationship of disease features is important. Although other methods exist for studying tissue in 3D, using conventional histopathological features has significant advantages because it allows for conventional histopathological staining and interpretation techniques. Until now, its use has not been routine in research because of the technical difficulty in constructing 3D tissue models. We describe a novel system for 3D histological reconstruction, integrating whole-slide imaging (virtual slides), image serving, registration, and visualization into one user-friendly package. It produces high-resolution 3D reconstructions with minimal user interaction and can be used in a histopathological laboratory without input from computing specialists. It uses a novel method for slice-to-slice image registration using automatic registration algorithms custom designed for both virtual slides and histopathological images. This system has been applied to >300 separate 3D volumes from eight different tissue types, using a total of 5500 virtual slides comprising 1.45 TB of primary image data. Qualitative and quantitative metrics for the accuracy of 3D reconstruction are provided, with measured registration accuracy approaching 120 μm for a 1-cm piece of tissue. Both 3D tissue volumes and generated 3D models are presented for four demonstrator cases.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

Resistance of CD-1 and ogg1 DNA Repair–Deficient Mice to Thalidomide and Hydrolysis Product Embryopathies in Embryo Culture

Thalidomide (TD) displays remarkable species specificity, causing birth defects (teratogenesis) in humans and rabbits, but not rats or mice; yet, few determinants of species susceptibility have been identified. Also, certain mouse strains are susceptible to the embryopathic effects of some teratogens in embryo culture despite their resistance in vivo. Herein we show that CD-1 mouse embryos in culture are resistant to limb embryopathies caused by TD and two of its hydrolysis products, 2-phthalimidoglutaramic acid and 2-phthalimidoglutaric acid, although all three compounds cause these embryopathies in rabbit embryo culture. These results show that the resistance of CD-1 mice to TD teratogenesis is inherent to the embryo and is not dependent upon maternal factors, including differential in vivo exposure to the many hydrolysis products of TD. In utero TD exposure of rabbit but not mouse embryos elevates levels of the teratogenic oxidative DNA lesion 8-oxoguanine, which is repaired by oxoguanine glycosylase 1 (OGG1). However, DNA repair-deficient ogg1 knockout mice proved resistant to TD-initiated embryopathies in culture and teratogenesis in vivo, indicating that the resistance of mice is not due to a higher level of DNA repair.

Endoglin Identifies the First Wave of Hematopoietic Progenitors During Embryogenesis.

AbstractAbstract 1600Endoglin (Eng), an ancillary receptor for several members of the Transforming Growth Factor (TGF)-beta superfamily, plays a critical role in early development. Eng-/- embryos dye around 10.5 dpc due primarily to vascular and cardiac abnormalities. Yolk sacs (YS) of 9.5 dpc Eng-/- embryos present abnormal vasculature and anemia. However, the mechanism by which endoglin leads to an anemic phenotype is unclear. Using in vitro differentiation of Eng-/- ES cells, we have previously demonstrated that endoglin is required for proper hemangioblast and primitive hematopoietic development. To test the hypothesis that endoglin might have a direct effect on blood formation, here we investigated the role of endoglin during hematopoiesis in vivo. We first evaluated YS from 9.5 dpc Eng-/- embryos and observed significantly reduced numbers of hematopoietic colony-forming cells (CFCs), in particular GEMMs and BFU-Es, when compared to both Eng+/− and Eng+/+ embryos. Real-time PCR analysis revealed decreased expression of embryonic globin, Gata-1, and SCL in the mutant YS. We then investigated the function of endoglin in wild-type mice by sorting the endoglin positive and negative population from E7.5 to 9.5 CD1 mouse embryos. When cultured on the stromal cell line OP9 in the presence of hematopoietic cytokines, only cell cultures derived from the Eng+ fraction gave rise to hematopoietic progenitors, as evidenced by colony assays and FACS analyses. Engneg cells did not produce any hematopoietic colonies. We then separated cells into 4 fractions using Eng and Flk-1, and found that from E7.5-9.5, the hematopoietic progenitor activity resided in the Eng+Flk-1+ double-positive fraction. FACS and microarray analyses of the 4 fractions in E7.5 embryos demonstrated that the Eng+Flk-1+ fraction highly expresses both endothelial and hematopoietic markers, including VE-Cadherin, Tie-2, PECAM1, Gata-1, SCL, and Lmo2, indicative of hemogenic endothelial potential. Taken together, our findings demonstrate that endoglin marks the first wave of hematopoietic progenitors during development, pointing to a defined role for endoglin in the specification of YS hemogenic endothelium as early as E7.5, in addition to its well defined role in vascular branching and remodeling. Disclosures:No relevant conflicts of interest to declare.

Ascorbic acid reverses valproic acid-induced inhibition of hoxa2 and maintains glutathione homeostasis in mouse embryos in culture.

Valproic acid (VPA) has been shown to cause neural tube defects in humans and mice, but its mechanism of action has not been elucidated. We hypothesize that alterations in embryonic antioxidant status and Hoxa2 gene expression play an important role in VPA-induced teratogenesis. A whole embryo culture system was applied to explore the effects of VPA on total glutathione, on glutathione in its oxidized (GSSG) and reduced (GSH) forms [GSSG/GSH ratio] and on Hoxa2 expression in cultured CD-1 mouse embryos during their critical period of organogenesis. Our results show that VPA can (1) induce embryo malformations including neural tube defects, abnormal flexion, yolk sac circulation defects, somite defects, and craniofacial deformities such as fusion of the first and second arches, and (2) alter glutathione homeostasis of embryos through an increase in embryonic GSSG/GSH ratio and a decrease in total GSH content in embryos. Western blot analysis and quantitative real-time RT-PCR show that VPA can inhibit Hoxa2 expression in cultured embryos at both the protein and mRNA level, respectively. The presence of ascorbic acid in the culture media was effective in protecting embryos against oxidative stress induced by VPA and prevented VPA-induced inhibition of Hoxa2 gene expression. Hoxa2 null mutant embryos do not exhibit altered glutathione homeostasis, indicating that inhibition of Hoxa2 is downstream of VPA-induced oxidative stress. These results are first to suggest VPA may, in part, exert its teratogenicity through alteration of the embryonic antioxidant status and inhibition of Hoxa2 gene expression and that ascorbic acid can protect embryos from VPA-induced oxidative stress.

A method to quantify apoptosis in mouse embryos using flow cytometry

Cell proliferation, differentiation and apoptosis play a major role in morphogenesis and organogenesis of the developing embryo. Teratogens can cause alteration in apoptotic patterns in the developing embryo which may lead to fetal malformations; however, no studies have quantified apoptosis in early embryos. We developed a protocol to quantify apoptosis in CD‐1 mouse embryos using flow cytometry. Valproic acid (VA) and diabetes mellitus were then used to induce neural tube defects and cardiac malformations respectively to evaluate apoptotic changes induced by known teratogens. Apoptotic levels were quantified in head regions of VA exposed embryos and hearts of hyperglycemic fetuses. Embryonic cells were disassociated enzymatically followed by differential staining with Annexin‐V and propidium iodide to differentiate apoptotic, dead and live cells. Apoptosis levels increased in the head regions of VA exposed embryos (18.1± 2.2 %) than the controls (12.7 ± 1.9 %). Increased levels were also observed in the hearts of hyperglycemic fetuses compared to controls. TUNEL assay, used to localize apoptotic regions, showed increased apoptosis along the open neural folds of the VA exposed embryos. This is the first report using flow cytometry to evaluate apoptosis in early embryos, and the results indicate this may be a useful method to quantify relative changes in apoptotic levels in teratogen exposure.Grant Funding SourceNIH grant K01RR16241‐01e

Secretion of stem cell factor and granulocyte–macrophage colony-stimulating factor by mouse embryos in culture: influence of group culture

Previous studies showed that the addition of a growth factor to the culture medium could modulate embryo development. The possible secretion of different factors to the culture medium by the embryo itself, however, has been poorly evaluated. The present study was designed to investigate: (1) the influence of single or group culture on the development of 2-cell mouse embryos (strain CD-1) to the blastocyst stage; (2) the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) into the culture medium by the embryo; and (3) the levels of GM-CSF and SCF in the culture medium from both single and group embryos. Two-cell CD-1 mouse embryos were cultured for 96 h singly or in groups of five embryos per drop. GM-CSF and SCF were assayed by ELISA in the complete culture medium. It was found that embryos cultured in groups gave a higher percentage of total blastocyst formation and hatched blastocyst when compared with single embryo culture. The mouse embryos secreted GM-CSF and SCF to the culture medium. The concentration of these cytokines is significantly higher in the group cultures than the level found in single cultures. In conclusion, mouse embryos in culture secrete GM-CSF and SCF to the culture medium and the concentration of these cytokines increases during communal culture. These factors may be operating in both autocrine and paracrine pathways to modulate embryo development during in vitro culture.