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.

Transcriptome-Wide Regulation of Key Developmental Pathways in the Mouse Neural Tube by Prenatal Alcohol Exposure.

BACKGROUND: Early gestational alcohol exposure is associated with severe craniofacial and CNS dysmorphologies and behavioral abnormalities during adolescence and adulthood. Alcohol exposure during the formation of the neural tube (gestational day [GD] 8 to 10 in mice; equivalent to4th week of human pregnancy) disrupts development of ventral midline brain structures such as the pituitary, septum, and ventricles. This study identifies transcriptomic changes in the rostroventral neural tube (RVNT), the region of the neural tube that gives rise to the midline structures sensitive to alcohol exposure during neurulation. METHODS: Female C57BL/6J mice were administered 2 doses of alcohol (2.9 g/kg) or vehicle 4 hours apart on GD 9.0. The RVNTs of embryos were collected 6 or 24 hours after the first dose and processed for RNA-seq. RESULTS: Six hours following GD 9.0 alcohol exposure (GD 9.25), over 2,300 genes in the RVNT were determined to be differentially regulated by alcohol. Enrichment analysis determined that PAE affected pathways related to cell proliferation, p53 signaling, ribosome biogenesis, and immune activation. In addition, over 100 genes involved in primary cilia formation and function and regulation of morphogenic pathways were altered 6 hours after alcohol exposure. The changes to gene expression were largely transient, as only 91 genes identified as differentially regulated by prenatal alcohol at GD 10 (24 hours postexposure). Functionally, the differentially regulated genes at GD 10 were related to organogenesis and cell migration. CONCLUSIONS: These data give a comprehensive view of the changing landscape of the embryonic transcriptome networks in regions of the neural tube that give rise to brain structures impacted by a neurulation-stage alcohol exposure. Identification of gene networks dysregulated by alcohol will help elucidate the pathogenic mechanisms of alcohol's actions.

Modulation of nuclear factor-κB signaling and reduction of neural tube defects by quercetin-3-glucoside in embryos of diabetic mice.

BACKGROUND: Diabetes mellitus in early pregnancy increases the risk of birth defects in infants. Maternal hyperglycemia stimulates the expression of nitric oxide synthase 2, which can be regulated by transcription factors of the nuclear factor-κB family. Increases in reactive nitrogen species generate intracellular stress conditions, including nitrosative, oxidative, and endoplasmic reticulum stresses, and trigger programmed cell death (or apoptosis) in the neural folds, resulting in neural tube defects in the embryo. Inhibiting nitric oxide synthase 2 can reduce neural tube defects; however, the underlying mechanisms require further delineation. Targeting nitric oxide synthase 2 and associated nitrosative stress using naturally occurring phytochemicals is a potential approach to preventing birth defects in diabetic pregnancies. OBJECTIVE: This study aims to investigate the effect of quercetin-3-glucoside, a naturally occurring polyphenol flavonoid, in reducing maternal diabetes-induced neural tube defects in an animal model, and to delineate the molecular mechanisms underlying quercetin-3-glucoside action in regulating nitric oxide synthase 2 expression. STUDY DESIGN: Female mice (C57BL/6) were induced to develop diabetes using streptozotocin before pregnancy. Diabetic pregnant mice were administered quercetin-3-glucoside (100 mg/kg) daily via gavage feeding, introduction of drug to the stomach directly via a feeding needle, during neurulation from embryonic day 6.5-9.5. After treatment at embryonic day 10.5, embryos were collected and examined for the presence of neural tube defects and apoptosis in the neural tube. Expression of nitric oxide synthase 2 and superoxide dismutase 1 (an antioxidative enzyme) was quantified using Western blot assay. Nitrosative, oxidative, and endoplasmic reticulum stress conditions were assessed using specific biomarkers. Expression and posttranslational modification of factors in the nuclear factor-κB system were investigated. RESULTS: Treatment with quercetin-3-glucoside (suspended in water) significantly decreased neural tube defect rate and apoptosis in the embryos of diabetic mice, compared with those in the water-treated diabetic group (3.1% vs. 24.7%; P < .001). Quercetin-3-glucoside decreased the expression of nitric oxide synthase 2 and nitrosative stress (P < .05). It also increased the levels of superoxide dismutase 1 (P < .05), further increasing the antioxidative capacity of the cells. Quercetin-3-glucoside treatment also alleviated of endoplasmic reticulum stress in the embryos of diabetic mice (P < .05). Quercetin-3-glucoside reduced the levels of p65 (P < .05), a member of the nuclear factor-κB transcription factor family, but augmented the levels of the inhibitor of κBα (P < .05), which suppresses p65 nuclear translocation. In association with these changes, the levels of inhibitor of κB kinase-α and inhibitor of κBα phosphorylation were elevated (P < .05). CONCLUSION: Quercetin-3-glucoside reduces the neural tube defects rate in the embryos of diabetic dams. Quercetin-3-glucoside suppresses nitric oxide synthase 2 and increases superoxide dismutase 1 expression, leading to alleviation of nitrosative, oxidative, and endoplasmic reticulum stress conditions. Quercetin-3-glucoside may regulate the expression of nitric oxide synthase 2 via modulating the nuclear factor-κB transcription regulation system. Quercetin-3-glucoside, a naturally occurring polyphenol that has high bioavailability and low toxicity, is a promising candidate agent to prevent birth defects in diabetic pregnancies.

The enduring impact of neurulation stage alcohol exposure: A combined behavioral and structural neuroimaging study in adult male and female C57BL/6J mice.

Prenatal alcohol exposure (PAE) can cause behavioral and brain alterations over the lifespan. In animal models, these effects can occur following PAE confined to critical developmental periods, equivalent to the third and fourth weeks of human gestation, before pregnancy is usually recognized. The current study focuses on PAE during early neurulation and examines the behavioral and brain structural consequences that appear in adulthood. On gestational day 8 C57BL/6J dams received two alcohol (2.8g/kg, i.p), or vehicle, administrations, four hours apart. Male and female offspring were reared to adulthood and examined for performance on the elevated plus maze, rotarod, open field, Morris water maze, acoustic startle, social preference (i.e. three-chambered social approach test), and the hot plate. A subset of these mice was later evaluated using magnetic resonance imaging to detect changes in regional brain volumes and shapes. In males, PAE increased exploratory behaviors on the elevated plus maze and in the open field; these changes were associated with increased fractional anisotropy in the anterior commissure. In females, PAE reduced social preference and the startle response, and decreased cerebral cortex and brain stem volumes. Vehicle-treated females had larger pituitaries than did vehicle-treated males, but PAE attenuated this sex difference. In males, pituitary size correlated with open field activity, while in females, pituitary size correlated with social activity. These findings indicate that early neurulation PAE causes sex specific behavioral and brain changes in adulthood. Changes in the pituitary suggest that this structure is especially vulnerable to neurulation stage PAE.

Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation.

Folate supplementation prevents up to 70% of neural tube defects (NTDs), which result from a failure of neural tube closure during embryogenesis. The elucidation of the mechanisms underlying folate action has been challenging. This study introduces Xenopus laevis as a model to determine the cellular and molecular mechanisms involved in folate action during neural tube formation. We show that knockdown of folate receptor 1 (Folr1; also known as FRα) impairs neural tube formation and leads to NTDs. Folr1 knockdown in neural plate cells only is necessary and sufficient to induce NTDs. Folr1-deficient neural plate cells fail to constrict, resulting in widening of the neural plate midline and defective neural tube closure. Pharmacological inhibition of folate action by methotrexate during neurulation induces NTDs by inhibiting folate interaction with its uptake systems. Our findings support a model in which the folate receptor interacts with cell adhesion molecules, thus regulating the apical cell membrane remodeling and cytoskeletal dynamics necessary for neural plate folding. Further studies in this organism could unveil novel cellular and molecular events mediated by folate and lead to new ways of preventing NTDs.

Metabolite profiling of whole murine embryos reveals metabolic perturbations associated with maternal valproate-induced neural tube closure defects.

BACKGROUND: Valproic acid (VPA) is prescribed therapeutically for multiple conditions, including epilepsy. When taken during pregnancy, VPA is teratogenic, increasing the risk of several birth and developmental defects including neural tube defects (NTDs). The mechanism by which VPA causes NTDs remains controversial and how VPA interacts with folic acid (FA), a vitamin commonly recommended for the prevention of NTDs, remains uncertain. We sought to address both questions by applying untargeted metabolite profiling analysis to neural tube closure (NTC) stage mouse embryos. METHODS: Pregnant SWV dams on either a 2 ppm or 10 ppm FA supplemented diet were injected with a single dose of VPA on gestational day E8.5. On day E9.5, the mouse embryos were collected and evaluated for NTC status. Liquid chromatography coupled to mass spectrometry metabolomics analysis was performed to compare metabolite profiles of NTD-affected VPA-exposed whole mouse embryos with profiles from embryos that underwent normal NTC from control dams. RESULTS: NTDs were observed in all embryos from VPA-treated dams and penetrance was not diminished by dietary FA supplementation. The most profound metabolic perturbations were found in the 10ppm FA VPA-exposed mouse embryos, compared with the other three treatment groups. Affected metabolites included amino acids, nucleobases and related phosphorylated nucleotides, lipids, and carnitines. CONCLUSION: Maternal VPA treatment markedly perturbed purine and pyrimidine metabolism in E9.5 embryos. In combination with a high FA diet, VPA treatment resulted in gross metabolic changes, likely caused by a multiplicity of mechanisms, including an apparent disruption of mitochondrial beta-oxidation. Birth Defects Research 109:106-119, 2017. © 2016 Wiley Periodicals, Inc.

Acute alcohol exposure during neurulation: Behavioral and brain structural consequences in adolescent C57BL/6J mice.

Prenatal alcohol exposure (PAE) can induce physical malformations and behavioral abnormalities that depend in part on thedevelopmental timing of alcohol exposure. The current studies employed a mouse FASD model to characterize the long-term behavioral and brain structural consequences of a binge-like alcohol exposure during neurulation; a first-trimester stage when women are typically unaware that they are pregnant. Time-mated C57BL/6J female mice were administered two alcohol doses (2.8g/kg, four hours apart) or vehicle starting at gestational day 8.0. Male and female adolescent offspring (postnatal day 28-45) were then examined for motor activity (open field and elevated plus maze), coordination (rotarod), spatial learning and memory (Morris water maze), sensory motor gating (acoustic startle and prepulse inhibition), sociability (three-chambered social test), and nociceptive responses (hot plate). Regional brain volumes and shapes were determined using magnetic resonance imaging. In males, PAE increased activity on the elevated plus maze and reduced social novelty preference, while in females PAE increased exploratory behavior in the open field and transiently impaired rotarod performance. In both males and females, PAE modestly impaired Morris water maze performance and decreased the latency to respond on the hot plate. There were no brain volume differences; however, significant shape differences were found in the cerebellum, hypothalamus, striatum, and corpus callosum. These results demonstrate that alcohol exposure during neurulation can have functional consequences into adolescence, even in the absence of significant brain regional volumetric changes. However, PAE-induced regional shape changes provide evidence for persistent brain alterations and suggest alternative clinical diagnostic markers.

Mono-2-ethylhexyl phthalate disrupts neurulation and modifies the embryonic redox environment and gene expression.

Mono-2-ethylhexl phthalate (MEHP) is the primary metabolite of di-2-ethylhexyl phthalate (DEHP), a ubiquitous contaminant in plastics. This study sought to determine how structural defects caused by MEHP in mouse whole embryo culture were related to temporal and spatial patterns of redox state and gene expression. MEHP reduced morphology scores along with increased incidence of neural tube defects. Glutathione (GSH) and cysteine (Cys) concentrations fluctuated spatially and temporally in embryo (EMB) and visceral yolk sac (VYS) across the 24h culture. Redox potentials (Eh) for GSSG/GSH were increased by MEHP in EMB (12h) but not in VYS. CySS/CyS Eh in EMB and VYS were significantly increased at 3h and 24h, respectively. Gene expression at 6h showed that MEHP induced selective alterations in EMB and VYS for oxidative phosphorylation and energy metabolism pathways. Overall, MEHP affects neurulation, alters Eh, and spatially alters the expression of metabolic genes in the early organogenesis-stage mouse conceptus.

Microtubule-associated protein 1b is required for shaping the neural tube.

BACKGROUND: Shaping of the neural tube, the precursor of the brain and spinal cord, involves narrowing and elongation of the neural tissue, concomitantly with other morphogenetic changes that contribue to this process. In zebrafish, medial displacement of neural cells (neural convergence or NC), which drives the infolding and narrowing of the neural ectoderm, is mediated by polarized migration and cell elongation towards the dorsal midline. Failure to undergo proper NC results in severe neural tube defects, yet the molecular underpinnings of this process remain poorly understood. RESULTS: We investigated here the role of the microtubule (MT) cytoskeleton in mediating NC in zebrafish embryos using the MT destabilizing and hyperstabilizing drugs nocodazole and paclitaxel respectively. We found that MTs undergo major changes in organization and stability during neurulation and are required for the timely completion of NC by promoting cell elongation and polarity. We next examined the role of Microtubule-associated protein 1B (Map1b), previously shown to promote MT dynamicity in axons. map1b is expressed earlier than previously reported, in the developing neural tube and underlying mesoderm. Loss of Map1b function using morpholinos (MOs) or δMap1b (encoding a truncated Map1b protein product) resulted in delayed NC and duplication of the neural tube, a defect associated with impaired NC. We observed a loss of stable MTs in these embryos that is likely to contribute to the NC defect. Lastly, we found that Map1b mediates cell elongation in a cell autonomous manner and polarized protrusive activity, two cell behaviors that underlie NC and are MT-dependent. CONCLUSIONS: Together, these data highlight the importance of MTs in the early morphogenetic movements that shape the neural tube and reveal a novel role for the MT regulator Map1b in mediating cell elongation and polarized cell movement in neural progenitor cells.

Dose-dependent teratogenicity of the synthetic cannabinoid CP-55,940 in mice.

Potent synthetic cannabinoids (SCBs) are illegally distributed drugs of abuse that are frequently consumed in spite of their adverse consequences. This study was designed to determine if the toxicity observed in adults also extends to the prenatal period by examining the developmental toxicity/teratogenicity of one of these SCBs, CP-55,940, in a mammalian model. First, immunohistochemistry was employed for cannabinoid receptor 1 (CB1) localization within gestational day (GD) 8 mouse embryos; this receptor was identified in the cranial neural plate, suggesting that the endogenous cannabinoid system may be involved in normal development. Based on this information and on previous avian teratogenicity studies, the current investigation focused on cannabinoid exposure during neurulation. The treatment paradigm involved acute i.p. administration of vehicle, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0mg/kg CP-55,940 to time-mated C57Bl/6J mice on their 8th day of pregnancy (n>10 litters per treatment group). On GD 17, litters were harvested and examined for numbers of live, dead, or resorbed fetuses, as well as for fetal weight, length, and gross morphological abnormalities. No effect on litter size, fetal weight, or crown rump length was seen at any of the CP-55,940 dosages tested. Major malformations involving the craniofacies and/or eyes were noted in all drug-treated groups. Selected fetuses with craniofacial malformations were histologically sectioned and stained, allowing investigation of brain anomalies. Observed craniofacial, ocular, and brain abnormalities in drug-treated fetuses included lateral and median facial clefts, cleft palate, microphthalmia, iridial coloboma, anophthalmia, exencephaly, holoprosencephaly, and cortical dysplasia. With the most commonly observed defects involving the eyes, the incidence and severity of readily identifiable ocular malformations were utilized as a basis for dose-response analyses. Ocular malformation ratings revealed dose-dependent CP-55,940 teratogenicity within the full range of dosages tested. While examination of additional critical periods and in depth mechanistic studies is warranted, the results of this investigation clearly show the dose-dependent teratogenicity of this SCB.

Low and moderate prenatal ethanol exposures of mice during gastrulation or neurulation delays neurobehavioral development.

Human and animal studies show significant delays in neurobehavioral development in offspring after prolonged prenatal exposure to moderate and high ethanol doses resulting in high blood alcohol concentration (BECs). However, none have investigated the effects of lower ethanol doses given acutely during specific developmental time periods. Here, we sought to create a mouse model for modest and circumscribed human drinking during the 3rd and 4th weeks of pregnancy. We acutely treated mice during embryo gastrulation on gestational day (GD) 7 or neurulation on GD8 with a low or moderate ethanol dose given via gavage that resulted in BECs of 107 and 177 mg/dl, respectively. We assessed neonatal physical development (pinnae unfolding, and eye opening); weight gain from postnatal day (PD) 3-65; and neurobehavioral maturation (pivoting, walking, cliff aversion, surface righting, vertical screen grasp, and rope balance) from PD3 to 17. We used a multiple linear regression model to determine the effects of dose, sex, day of treatment and birth in animals dosed during gastrulation or neurulation, relative to their vehicle controls. We found that ethanol exposure during both time points (GD7 and GD8) resulted in some delays of physical development and significant sensorimotor delays of pivoting, walking, and thick rope balance, as well as additional significant delays in cliff aversion and surface righting after GD8 treatment. We also found that treatment with the low ethanol dose more frequently affected neurobehavioral development of the surviving pups than treatment with the moderate ethanol dose, possibly due to a loss of severely affected offspring. Finally, mice born prematurely were delayed in their physical and sensorimotor development. Importantly, we showed that brief exposure to low dose ethanol, if administered during vulnerable periods of neuroanatomical development, results in significant neurobehavioral delays in neonatal mice. We thus expand concerns about alcohol consumption during the 3rd and 4th weeks of human pregnancy to include occasional light to moderate drinking.

Early maternal alcohol consumption alters hippocampal DNA methylation, gene expression and volume in a mouse model.

The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first 8 days of gestation (GD 0.5-8.5). Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P) 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60): we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in different tissue types later in life.

Dorsoventral patterning of the Xenopus eye involves differential temporal changes in the response of optic stalk and retinal progenitors to Hh signalling.

BACKGROUND: Hedgehog (Hh) signals are instrumental to the dorsoventral patterning of the vertebrate eye, promoting optic stalk and ventral retinal fates and repressing dorsal retinal identity. There has been limited analysis, however, of the critical window during which Hh molecules control eye polarity and of the temporal changes in the responsiveness of eye cells to these signals. RESULTS: In this study, we used pharmacological and molecular tools to perform stage-specific manipulations of Hh signalling in the developing Xenopus eye. In gain-of-function experiments, most of the eye was sensitive to ventralization when the Hh pathway was activated starting from gastrula/neurula stages. During optic vesicle stages, the dorsal eye became resistant to Hh-dependent ventralization, but this pathway could partially upregulate optic stalk markers within the retina. In loss-of-function assays, inhibition of Hh signalling starting from neurula stages caused expansion of the dorsal retina at the expense of the ventral retina and the optic stalk, while the effects of Hh inhibition during optic vesicle stages were limited to the reduction of optic stalk size. CONCLUSIONS: Our results suggest the existence of two competence windows during which the Hh pathway differentially controls patterning of the eye region. In the first window, between the neural plate and the optic vesicle stages, Hh signalling exerts a global influence on eye dorsoventral polarity, contributing to the specification of optic stalk, ventral retina and dorsal retinal domains. In the second window, between optic vesicle and optic cup stages, this pathway plays a more limited role in the maintenance of the optic stalk domain. We speculate that this temporal regulation is important to coordinate dorsoventral patterning with morphogenesis and differentiation processes during eye development.

A rat toxicogenomics study with the calcium sensitizer EMD82571 reveals a pleiotropic cause of teratogenicity.

The calcium sensitizer and PDEIII inhibitor EMD82571 caused exencephaly, micrognathia, agnathia and facial cleft in 58% of fetuses. In pursue of mechanisms and to define adverse outcome pathways pregnant Wistar rats were dosed daily with either EMD82571 (50 or 150mg/kg/day) or retinoic acid (12mg/kg/day) on gestational days 6-11 and 6-17, respectively. Hypothesis driven and whole genome microarray experiments were performed with whole embryo, maternal liver, embryonic liver and malformed bone at gestational days 12 and 20. This revealed regulation of genes critically involved in osteogenesis, odontogenesis, differentiation and development and extracellular matrix. Importantly, repression of osteocalcin and members of TGF-ß/BMP signaling hampered osteo- and odontogenesis. Furthermore, EMD82571 impaired neurulation by inhibiting mid hinge point formation to cause neural tube defects. Taken collectively, a molecular rationale for the observed teratogenicity induced by EMD82571 is presented that links molecular initiating events with AOPs.

Vinyl chloride monomer (VCM) induces high occurrence of neural tube defects in embryonic mouse brain during neurulation.

The aim of this study was to explore the direct embryonic teratogenicity of vinyl chloride monomer (VCM), especially the toxic effects on the early development of the nervous system and its underlying mechanisms. Pregnant mice at embryonic day 6.5 (E6.5) were injected with different doses of VCM (200, 400 and 600 mg/kg) and embryos were harvested at E10.5. Our results showed that doses higher than 400 mg/kg of VCM increased the incidence of malformed embryos, especially the neural tube defects (NTDs). In addition, high-dose of VCM decreased mitotic figure counts in the neuroepithelium and enhanced the percentage of cells in G0/G1 phase, while they were reduced in S phase. The more VCM was injected into mice, the fewer positive PCNA cells were seen and the more positive TUNEL cells were observed in the neuroepithelium. Moreover, significant increases in the levels of caspase-3 protein were observed in NTD embryos. Our results demonstrate that during early pregnancy, exposure to doses higher than 400 mg/kg of VCM increases the incidence of malformations and particularly the rate of NTDs. High-dose of VCM inhibits the proliferation of neural cells and induces cell apoptosis, leading to an imbalance in the ratio of proliferation and apoptosis. Meanwhile, the apoptosis of neuroepithelial cells might be accelerated by the activation of the caspase-3 pathway, and it might be a reason for NTDs.

Role of methotrexate exposure in apoptosis and proliferation during early neurulation.

Apoptosis and proliferation play important roles in embryonic development and are required for neural tube closure. The antifolate drug methotrexate (MTX) induces folate dysmetabolism by inhibition of dihydrofolate reductase and causes abnormal apoptosis and proliferation. In this study, we established an animal model of neural tube defects (NTDs) using MTX to investigate the role of apoptosis and proliferation in NTDs caused by folate deficiency. Differential gene expressions were studied by microarray and reverse transcription-polymerase chain reaction in the NTD animal model. Results showed that 30.8% of NTDs were caused by using MTX in treatment regimens. Microarray indicated that 166 genes were significantly different between the control and NTD mice, including four apoptosis-related genes (Endog, Trp53, Casp3, Bax) and three proliferation-related genes (Ptch1, Pla2g4a, Foxg1). Levels of Endog, Trp53, Casp3, Bax (fold change>1.5) were upregulated but Ptch1, Pla2g4a, Foxg1 (fold change<0.67) were downregulated (P<0.05). These results were confirmed by reverse transcription-polymerase chain reaction. TUNEL, immunohistochemical assays and Western blot were further used to detect apoptosis and proliferation in the NTD animal model. It was found that apoptosis in neuroepithelial cells was increased as determined by TUNEL (P<0.05). Expressions of caspase-3 were significantly enhanced (P<0.05) but expressions of phosphohistone H3 were greatly decreased (P<0.05). These results concluded that MTX caused a folate and folate-associated dysmetabolism, and further induced abnormal apoptosis and proliferation, which may play a critical role in the occurrence of NTDs caused by folate deficiency.

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40-60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca(2+)-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both-indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.

c-Jun NH2-terminal kinase 1/2 and endoplasmic reticulum stress as interdependent and reciprocal causation in diabetic embryopathy.

Embryos exposed to high glucose exhibit aberrant maturational and cytoarchitectural cellular changes, implicating cellular organelle stress in diabetic embryopathy. c-Jun-N-terminal kinase 1/2 (JNK1/2) activation is a causal event in maternal diabetes-induced neural tube defects (NTD). However, the relationship between JNK1/2 activation and endoplasmic reticulum (ER) stress in diabetic embryopathy has never been explored. We found that maternal diabetes significantly increased ER stress markers and induced swollen/enlarged ER lumens in embryonic neuroepithelial cells during neurulation. Deletion of either jnk1 or jnk2 gene diminished hyperglycemia-increased ER stress markers and ER chaperone gene expression. In embryos cultured under high-glucose conditions (20 mmol/L), the use of 4-phenylbutyric acid (4-PBA), an ER chemical chaperone, diminished ER stress markers and abolished the activation of JNK1/2 and its downstream transcription factors, caspase 3 and caspase 8, and Sox1 neural progenitor apoptosis. Consequently, both 1 and 2 mmol/L 4-PBA significantly ameliorated high glucose-induced NTD. We conclude that hyperglycemia induces ER stress, which is responsible for the proapoptotic JNK1/2 pathway activation, apoptosis, and NTD induction. Suppressing JNK1/2 activation by either jnk1 or jnk2 gene deletion prevents ER stress. Thus, our study reveals a reciprocal causation of ER stress and JNK1/2 in mediating the teratogenicity of maternal diabetes.

Does the cranial mesenchyme contribute to neural fold elevation during neurulation?

The central nervous system is derived from the neural plate, which undergoes a series of complex morphogenetic events resulting in formation of the neural tube in a process known as neurulation. The cellular behaviors driving neurulation in the cranial region involve forces generated by the neural tissue itself as well as the surrounding epithelium and mesenchyme. Of interest, the cranial mesenchyme underlying the neural plate undergoes stereotypical rearrangements hypothesized to drive elevation of the neural folds. As the neural folds rise, the hyaluronate-rich extracellular matrix greatly expands resulting in increased space between individual cranial mesenchyme cells. Based on inhibitor studies, expansion of the extracellular matrix has been implicated in driving neural fold elevation; however, because the surrounding neural and epidermal ectoderm were also affected by inhibitor exposure, these studies are inconclusive. Similarly, treatment of neurulating embryos with teratogenic doses of retinoic acid results in altered organization of the cranial mesenchyme, but alterations in surrounding tissues are also observed. The strongest evidence for a critical role for the cranial mesenchyme in neural fold elevation comes from studies of genes expressed exclusively in the cranial mesenchyme that when mutated result in exencephaly associated with abnormal organization of the cranial mesenchyme. Twist is the best studied of these and is expressed in both the paraxial mesoderm and neural crest derived cranial mesenchyme. In this article, we review the evidence implicating the cranial mesenchyme in providing a driving force for neural fold elevation to evaluate whether there are sufficient data to support this hypothesis.

Simultaneous folate intake may prevent adverse effect of valproic acid on neurulating nervous system.

PURPOSE: The aim of this study is to elucidate the preventive effect of folic acid (FA) on teratogenic effects of valporic acid (VA) in early stage chick embryos on neural tube development. MATERIALS AND METHODS: One hundred and fifty specific pathogen-free (SPF) chick eggs were used to investigate the neurulation in five groups. Group A was the control group. Group B was injected 0.02 ml of saline (0.9% NaCl) and was used for sham group. VA (0.72 mg) in 0.02 ml saline was injected in Group C, and 0.342 mcg of FA in 0.02 ml NaCl were administered to the embryos in Group D. VA (0.72 mg) + 0.342 mcg of FA in 0.02 ml saline were administered simultaneously to the eggs in Group E. At the end of 72 h, all embryos were extracted from eggs and were fixed, and for histological analyses hematoxylin and eosine was used, for detection of apoptotic cells terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL) was used and for distribution of P53, bcl-2 and caspase-3, caspase-6, caspase-8 and caspase-9 immunoperoxidase techniques were used. RESULTS: While there were no neural tube defects in the embryos of groups A, B and D, eight embryos died in group C and there were 12 embryos with retarded embryological development. In contrast to that, no death was observed in group E, but only eight embryos were detected with maldevelopmental delay stage. CONCLUSION: These results suggested that VA may induce apoptotic mechanisms but not through the p53 pathway. In addition, FA effectively prevents the teratogenic influence of VA on chick embryo at neurulation stages by stopping cascade of apoptosis before caspase 3 expression.