Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

CIC missense variants contribute to susceptibility for spina bifida.

Neural tube defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to the cerebral folate deficiency syndrome by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole-genome sequencing (WGS) data of 140 isolated spina bifida cases and identified eight missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased the FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants downregulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD.

FKBP8 variants are risk factors for spina bifida.

Neural tube defects (NTDs) are a group of severe congenital malformations caused by a failure of neural tube closure during early embryonic development. Although extensively investigated, the genetic etiology of NTDs remains poorly understood. FKBP8 is critical for proper mammalian neural tube closure. Fkbp8-/- mouse embryos showed posterior NTDs consistent with a diagnosis of spina bifida (SB). To date, no publication has reported any association between FKBP8 and human NTDs. Using Sanger sequencing on genomic DNA samples from 472 SB and 565 control samples, we identified five rare (MAF ≤ 0.001) deleterious variants in SB patients, while no rare deleterious variant was identified in the controls (P = 0.0191). p.Glu140* affected FKBP8 localization to the mitochondria and created a truncated form of the FKBP8 protein, thus impairing its interaction with BCL2 and ultimately leading to an increase in cellular apoptosis. p.Ser3Leu, p.Lys315Asn and p.Ala292Ser variants decreased FKBP8 protein level. p.Lys315Asn further increased the cellular apoptosis. RNA sequencing on anterior and posterior tissues isolated from Fkbp8-/- and wildtype mice at E9.5 and E10.5 showed that Fkbp8-/- embryos have an abnormal expression profile within tissues harvested at posterior sites, thus leading to a posterior NTD. Moreover, we found that Fkbp8 knockout mouse embryos have abnormal expression of Wnt3a and Nkx2.9 during the early stage of neural tube development, perhaps also contributing to caudal specific NTDs. These findings provide evidence that functional variants of FKBP8 are risk factors for SB, which may involve a novel mechanism by which Fkbp8 mutations specifically cause SB in mice.

CIC de novo loss of function variants contribute to cerebral folate deficiency by downregulating FOLR1 expression.

Background Cerebral folate deficiency (CFD) syndrome is characterised by a low concentration of 5-methyltetrahydrofolate in cerebrospinal fluid, while folate levels in plasma and red blood cells are in the low normal range. Mutations in several folate pathway genes, including FOLR1 (folate receptor alpha, FRα), DHFR (dihydrofolate reductase) and PCFT (proton coupled folate transporter) have been previously identified in patients with CFD. Methods In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis on eight CFD trios and identified eight de novo mutations in seven trios. Results Notably, we found a de novo stop gain mutation in the capicua (CIC) gene. Using 48 sporadic CFD samples as a validation cohort, we identified three additional rare variants in CIC that are putatively deleterious mutations. Functional analysis indicates that CIC binds to an octameric sequence in the promoter regions of folate transport genes: FOLR1, PCFT and reduced folate carrier (Slc19A1; RFC1). The CIC nonsense variant (p.R353X) downregulated FOLR1 expression in HeLa cells as well as in the induced pluripotent stem cell (iPSCs) derived from the original CFD proband. Folate binding assay demonstrated that the p.R353X variant decreased cellular binding of folic acid in cells. Conclusion This study indicates that CIC loss of function variants can contribute to the genetic aetiology of CFD through regulating FOLR1 expression. Our study described the first mutations in a non-folate pathway gene that can contribute to the aetiology of CFD.

Dominant negative GPR161 rare variants are risk factors of human spina bifida.

Spina bifida (SB) is a complex disorder of failed neural tube closure during the first month of human gestation, with a suspected etiology involving multiple gene and environmental interactions. GPR161 is a ciliary G-protein coupled receptor that regulates Sonic Hedgehog (Shh) signaling. Gpr161 null and hypomorphic mutations cause neural tube defects (NTDs) in mouse models. Herein we show that several genes involved in Shh and Wnt signaling were differentially expressed in the Gpr161 null embryos using RNA-seq analysis. To determine whether there exists an association between GPR161 and SB in humans, we performed direct Sanger sequencing on the GPR161 gene in a cohort of 384 SB patients and 190 healthy controls. We identified six rare variants of GPR161 in six SB cases, of which two of the variants were novel and did not exist in any databases. Both of these variants were predicted to be damaging by SIFT and/or PolyPhen analysis. The novel GPR161 rare variants mislocalized to the primary cilia, dysregulated Shh and Wnt signaling and inhibited cell proliferation in vitro. Our results demonstrate that GPR161 mutations cause NTDs via dysregulation of Shh and Wnt signaling in mice, and novel rare variants of GPR161 can be risk factors for SB in humans.

Formate rescues neural tube defects caused by mutations in Slc25a32.

Periconceptional folic acid (FA) supplementation significantly reduces the prevalence of neural tube defects (NTDs). Unfortunately, some NTDs are FA resistant, and as such, NTDs remain a global public health concern. Previous studies have identified SLC25A32 as a mitochondrial folate transporter (MFT), which is capable of transferring tetrahydrofolate (THF) from cellular cytoplasm to the mitochondria in vitro. Herein, we show that gene trap inactivation of Slc25a32 (Mft) in mice induces NTDs that are folate (5-methyltetrahydrofolate, 5-mTHF) resistant yet are preventable by formate supplementation. Slc25a32gt/gt embryos die in utero with 100% penetrant cranial NTDs. 5-mTHF supplementation failed to promote normal neural tube closure (NTC) in mutant embryos, while formate supplementation enabled the majority (78%) of knockout embryos to complete NTC. A parallel genetic study in human subjects with NTDs identified biallelic loss of function SLC25A32 variants in a cranial NTD case. These data demonstrate that the loss of functional Slc25a32 results in cranial NTDs in mice and has also been observed in a human NTD patient.

The interaction of maternal diabetes with mutations that affect folate metabolism and how they affect the development of neural tube defects in mice.

BACKGROUND: Abnormalities in maternal folate and carbohydrate metabolism have both been shown to induce neural tube defects (NTDs) in humans and animal models. Nevertheless, how these two factors might interact in the development of NTDs remains unclear. RESULTS: In specific mouse models and embryo culture systems, we assessed the effects of combining maternal diabetes with mutations in genes involved in folate transport and metabolism (methylenetetrahydrofolate reductase [Mthfr] and folic acid receptor 1 [Folr1]). When maternal hyperglycemia is combined with alterations in folic acid metabolism, there appears to be an increase in the incidence of congenital malformations in the offspring, with NTDs representing the majority of the malformations detected. CONCLUSIONS: The teratogenic effects of diabetes during pregnancy are exacerbated when combined with altered embryonic folate metabolism.

Comparative teratogenicity analysis of valnoctamide, risperidone, and olanzapine in mice.

OBJECTIVES: Based on the recent findings from animal studies, it has been proposed that the therapeutic use of valnoctamide, an anxiolytic drug developed in the early 1960s, be extended to treat other neurological disorders such as epilepsy and bipolar disease. Given the scarcity of adequate data on its prenatal toxicity, a comparative teratogenicity study of valnoctamide and two of the most commonly used drugs to treat bipolar disorder, risperidone and olanzapine, was carried out in a mouse model system. METHODS: Pregnant dams were treated with the aforementioned three drugs at the dose levels calculated as an equal proportion of the respective LD50 values of these drugs. The main reproductive indices examined included the numbers of implantations and resorptions, viable and dead fetuses, and fetal gross, visceral and skeletal abnormalities. RESULTS: The outcomes of the present study indicated that olanzapine was the most teratogenic of the three drugs, inducing maternal-, embryo-, and fetotoxicity. Risperidone also exerted a significant prenatal toxicity, but its adverse effect was less pronounced than that induced by olanzapine. Valnoctamide did not show any teratogenic effect, even when used in relatively higher dosages than olanzapine and risperidone. The observed increased skeletal abnormalities in one of the valnoctamide treatment groups were nonspecific and, as such, signaled a modest developmental delay rather than an indication that the compound could induce structural malformations. CONCLUSIONS: Under our experimental conditions, valnoctamide demonstrated the lowest prenatal toxicity of the three tested drugs.

Pharmacodynamic and pharmacokinetic analysis of CNS-active constitutional isomers of valnoctamide and sec-butylpropylacetamide--Amide derivatives of valproic acid.

Valnoctamide (VCD) and sec-butylpropylacetamide (SPD) are CNS-active closely related amide derivatives of valproic acid with unique anticonvulsant activity. This study evaluated how small chemical changes affect the pharmacodynamics (PD; anticonvulsant activity and teratogenicity) and pharmacokinetics (PK) of three constitutional isomers of SPD [sec-butylisopropylacetamide (SID) and tert-butylisopropylacetamide (TID)] and of VCD [tert-butylethylacetamide (TED)]. The anticonvulsant activity of SID, TID, and TED was comparatively evaluated in several rodent anticonvulsant models. The PK-PD relationship of SID, TID, and TED was evaluated in rats, and their teratogenicity was evaluated in a mouse strain highly susceptible to teratogen-induced neural tube defects (NTDs). sec-Butylisopropylacetamide and TID have a similar PK profile to SPD which may contribute to their similar anticonvulsant activity. tert-Butylethylacetamide had a better PK profile than VCD (and SPD); however, this did not lead to a superior anticonvulsant activity. sec-Butylisopropylacetamide and TED did not cause NTDs at doses 4-7 times higher than their anticonvulsant ED50 values. In rats, SID, TID (ip), and TED exhibited a broad spectrum of anticonvulsant activity. However, combined anticonvulsant analysis in mice and rats shows SID as the most potent compound with similar activity to that of SPD, demonstrating that substitution of the isobutyl moiety in the SPD or VCD molecule by tert-butyl as well as a propyl-to-isopropyl replacement in the SPD molecule did not majorly affect the anticonvulsant activity.

Mthfr gene ablation enhances susceptibility to arsenic prenatal toxicity.

BACKGROUND: In utero exposure to arsenic is known to adversely affect reproductive outcomes. Evidence of arsenic teratogenicity varies widely and depends on individual genotypic differences in sensitivity to As. In this study, we investigated the potential interaction between 5,10-methylenetetrahydrofolate reductase (Mthfr) genotype and arsenic embryotoxicity using the Mthfr knockout mouse model. METHODS: Pregnant dams were treated with sodium arsenate, and reproductive outcomes including: implantation, resorption, congenital malformation and fetal birth weight were recorded at E18.5. RESULTS: When the dams in Mthfr(+/-)×Mthfr(+/-) matings were treated with 7.2 mg/kg As, the resorption rate increased to 43.4%, from a background frequency of 7.2%. The As treatment also induced external malformations (40.9%) and significantly lowered the average fetal birth weight among fetuses, without any obvious toxic effect on the dam. When comparing the pregnancy outcomes resulting from different mating scenarios (Mthfr(+/+)×Mthfr(+/-), Mthfr(+/-)×Mthfr(+/-) and Mthfr(-/-)×(Mthfr+/-)) and arsenic exposure; the resorption rate showed a linear relationship with the number of null alleles (0, 1 or 2) in the Mthfr dams. Fetuses from nullizygous dams had the highest rate of external malformations (43%) and lowest average birth weight. When comparing the outcomes of reciprocal matings (nullizygote×wild-type versus wild-type×nullizygote) after As treatment, the null dams showed significantly higher rates of resorptions and malformations, along with lower fetal birth weights. CONCLUSIONS: Maternal genotype contributes to the sensitivity of As embryotoxicity in the Mthfr mouse model. The fetal genotype, however, does not appear to affect the reproductive outcome after in utero As exposure.

The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system.

The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01-0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected.

Enantioselective pharmacodynamic and pharmacokinetic analysis of two chiral CNS-active carbamate derivatives of valproic acid.

OBJECTIVE: 2-Ethyl-3-methylbutyl-carbamate (EMC) and 2-isopropylpentyl-carbamate (IPC) are among the most potent anticonvulsant carbamate derivatives of valproic acid. EMC and IPC are chiral compounds. Consequently, the aim of the current study was to comparatively evaluate the pharmacokinetic (PK) and pharmacodynamic (PD anticonvulsant activity) profile of EMC and IPC individual enantiomers. METHODS: The anticonvulsant activity of EMC and IPC individual enantiomers was evaluated in several anticonvulsant rodent models including maximal electroshock (MES), 6 Hz psychomotor, subcutaneous (pentylenetetrazole) (scMet), and the pilocarpine-induced and soman-induced status epilepticus (SE). The PK-PD relationship of EMC and IPC individual enantiomers was evaluated following intraperitoneal administration (50 mg/kg) to rats. Induction of neural tube defects (NTDs) was evaluated in a mouse strain that was highly susceptible to teratogen-induced NTDs. RESULTS: In mice and rats, (2S)-EMC exhibited anticonvulsant activity similar to that of racemic EMC in the MES and scMet tests, whereas in the 6 Hz test, racemic EMC was more potent than its two individual enantiomers. Racemic EMC exhibited a potent activity in the soman-induced SE model when administered 5 and 20 min after seizure onset with median effective dose (ED50 ) values of 33 and 48 mg/kg, respectively. (2R)-IPC and (2S)-IPC exhibited ED50 values similar to those of racemic IPC in the mouse and rat MES and scMet models. (2R)-IPC had similar ED50 values on the 6 Hz tests. Racemic IPC had an ED50 value of 107 mg/kg in the pilocarpine-induced SE model when given 30 min after seizure onset. Racemic EMC and IPC and their enantiomers had similar clearance (3.8-5.5 L/h/kg) and short half-life (<1 h). EMC and its enantiomers did not cause NTDs at doses 3-10 times higher than their anticonvulsant ED50 values. SIGNIFICANCE: EMC and IPC did not exhibit enantioselective PK, a fact that may contribute to their nonenantioselective activity in any of the anticonvulsant models. The nonsignificant difference between racemic EMC and racemic IPC and their enantiomers, suggests that their wide spectrum of anticonvulsant activity is likely to be caused by multiple mechanisms of action.

Stereoselective anticonvulsant and pharmacokinetic analysis of valnoctamide, a CNS-active derivative of valproic acid with low teratogenic potential.

OBJECTIVE: Valnoctamide (VCD), a central nervous system (CNS)-active chiral constitutional isomer of valpromide, the corresponding amide of valproic acid (VPA), is currently undergoing phase IIb clinical trials in acute mania. VCD exhibits stereoselective pharmacokinetics (PK) in animals and humans. The current study comparatively evaluated the pharmacodynamics (PD; anticonvulsant activity and teratogenicity) and PK of the four individual stereoisomers of VCD. METHODS: The anticonvulsant activity of VCD individual stereoisomers was evaluated in several rodent anticonvulsant models including maximal electroshock, 6 Hz psychomotor, subcutaneous metrazol, and the pilocarpine-induced and soman-induced status epilepticus (SE). The PK-PD (anticonvulsant activity) relationship of VCD stereoisomers was evaluated following intraperitoneal administration (70 mg/kg) to rats. Induction of neural tube defects (NTDs) by VCD stereoisomers was evaluated in a mouse strain that was highly susceptible to teratogen-induced NTDs. RESULTS: VCD had a stereoselective PK, with (2S,3S)-VCD exhibiting the lowest clearance, and consequently a twice-higher plasma exposure than all other stereoisomers. Nervertheless, there was less stereoselectivity in VCD anticonvulsant activity and each stereoisomer had similar median effective dose (ED)50 values in most models. VCD stereoisomers (258 or 389 mg/kg) did not cause NTDs. These doses are 3-12 times higher than VCD anticonvulsant ED50 values. SIGNIFICANCE: VCD displayed stereoselective PK that did not lead to significant stereoselective activity in various anticonvulsant rodent models. If VCD exerted its broad-spectrum anticonvulsant activity using a single mechanism of action (MOA), it is likely that it would exhibit a stereoselective PD. The fact that there was no significant difference between racemic VCD and its individual stereoisomers suggests that VCD's anticonvulsant activity is due to multiple MOAs.

The novel linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse embryonic development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing primary cilia, the cell antenna acting as a signaling hub. Fuz, an effector of planar cell polarity (PCP) signaling, involves Shh signaling via cilia formation, while the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of these two genes is similar; however, their functional relations have not been previously explored. This study identified the genetic and biochemical link between Fuz and Gpr161 in mouse embryonic development. Fuz was genetically epistatic to Gpr161 via Shh signaling during mouse embryonic development. The FUZ biochemically interacted with GPR161, and Fuz regulated Gpr161 ciliary trafficking via ß-arrestin2. Our study suggested the novel Gpr161-Fuz axis that regulates Shh signaling during mouse embryonic development.

Dolutegravir-induced neural tube defects in mice are folate responsive.

OBJECTIVES: In 2018, the Botswana Tsepamo Study reported a nine-fold increased risk of neural tube defects in infants whose mothers were treated with dolutegravir (DTG) from the time of conception. As maternal folate supplementation and status is a well known modifier of neural tube defect (NTD) risk, we sought to evaluate birth outcomes in mice fed normal and low folic acid diets treated with DTG during pregnancy. DESIGN: DTG was evaluated for developmental toxicity using pregnant mice fed normal or low folic acid diet. METHODS: CD-1 mice were provided diet with normal (3 mg/kg) or low (0.3 mg/kg) folic acid. They were treated with water, a human therapeutic-equivalent dose, or supratherapeutic dose of DTG from mouse embryonic day E6.5 to E12.5. Pregnant dams were sacrificed at term (E18.5) and fetuses were inspected for gross, internal, and skeletal defects. RESULTS: Fetuses with exencephaly, an NTD, were present in both therapeutic human equivalent and supratherapeutic exposures in dams fed low folic acid diet. Cleft palates were also found under both folate conditions. CONCLUSIONS: Recommended dietary folic acid levels during mouse pregnancy ameliorate developmental defects that arise from DTG exposure. Since low folate status in mice exposed to DTG increases the risk for NTDs, it is possible that DTG exposures in people living with HIV with low folate status during pregnancy may explain, at least in part, the elevated NTD risk signal observed in Botswana. Based on these results, future studies should consider folate status as a modifier for DTG-associated NTD risk.

Antiepileptic drugs and pregnancy outcomes.

The treatment of epilepsy in women of reproductive age remains a clinical challenge. While most women with epilepsy (WWE) require anticonvulsant drugs for adequate control of their seizures, the teratogenicity associated with some antiepileptic drugs (AEDs) is a risk that needs to be carefully addressed. Antiepileptic medications are also used to treat an ever broadening range of medical conditions such as bipolar disorder, migraine prophylaxis, cancer, and neuropathic pain. Despite the fact that the majority of pregnancies of WWE who are receiving pharmacological treatment are normal, studies have demonstrated that the risk of having a pregnancy complicated by a major congenital malformation is doubled when comparing the risk of untreated pregnancies. Furthermore, when AEDs are used in polytherapy regimens, the risk is tripled, especially when valproic acid (VPA) is included. However, it should be noted that the risks are specific for each anticonvulsant drug. Some investigations have suggested that the risk of teratogenicity is increased in a dose-dependent manner. More recent studies have reported that in utero exposure to AEDs can have detrimental effects on the cognitive functions and language skills in later stages of life. In fact, the FDA just issued a safety announcement on the impact of VPA on cognition (Safety Announcement 6-30-2011). The purpose of this document is to review the most commonly used compounds in the treatment of WWE, and to provide information on the latest experimental and human epidemiological studies of the effects of AEDs in the exposed embryos.

Transcriptional analyses of two mouse models of spina bifida.

BACKGROUND: Spina bifida is one of the most common of all human structural birth defects. Despite considerable effort over several decades, the causes and mechanisms underlying this malformation remain poorly characterized. METHODS: To better understand the pathogenesis of this abnormality, we conducted a microarray study using Mouse Whole Genome Bioarrays which have ~36,000 gene targets, to compare gene expression profiles between two mouse models; CXL-Splotch and FKBP8(Gt(neo)) which express a similar spina bifida phenotype. We anticipated that there would be a collection of overlapping genes or shared genetic pathways at the molecular level indicative of a common mechanism underlying the pathogenesis of spina bifida during embryonic development. RESULTS: A total of 54 genes were determined to be differentially expressed (25 downregulated, 29 upregulated) in the FKBP8Gt((neo)) mouse embryos; whereas 73 genes were differentially expressed (56 downregulated, 17 upregulated) in the CXL-Splotch mouse relative to their wild-type controls. Remarkably, the only two genes that showed decreased expression in both mutants were v-ski sarcoma viral oncogene homolog (Ski), and Zic1, a transcription factor member of the zinc finger family. Confirmation analysis using quantitative real-time (qRT)-PCR indicated that only Zic1 was significantly decreased in both mutants. Gene ontology analysis revealed striking enrichment of genes associated with mesoderm and central nervous system development in the CXL-Splotch mutant embryos, whereas in the FKBP8(Gt(neo)) mutants, the genes involved in dorsal/ventral pattern formation, cell fate specification, and positive regulation of cell differentiation were most likely to be enriched. These results indicate that there are multiple pathways and gene networks perturbed in mouse embryos with shared phenotypes.

Embryonic Hypotaurine Levels Contribute to Strain-Dependent Susceptibility in Mouse Models of Valproate-Induced Neural Tube Defects.

Valproic acid (VPA, valproate, Depakote) is a commonly used anti-seizure medication (ASM) in the treatment of epilepsy and a variety of other neurological disorders. While VPA and other ASMs are efficacious for management of seizures, they also increase the risk for adverse pregnancy outcomes, including neural tube defects (NTDs). Thus, the utility of these drugs during pregnancy and in women of childbearing potential presents a continuing public health challenge. Elucidating the underlying genetic or metabolic risk factors for VPA-affected pregnancies may lead to development of non-teratogenic ASMs, novel prevention strategies, or more targeted methods for managing epileptic pregnancies. To address this challenge, we performed unbiased, whole embryo metabolomic screening of E8.5 mouse embryos from two inbred strains with differential susceptibility to VPA-induced NTDs. We identified metabolites of differential abundance between the two strains, both in response to VPA exposure and in the vehicle controls. Notable enriched pathways included lipid metabolism, carnitine metabolism, and several amino acid pathways, especially cysteine and methionine metabolism. There also was increased abundance of ω-oxidation products of VPA in the more NTD-sensitive strain, suggesting differential metabolism of the drug. Finally, we found significantly reduced levels of hypotaurine in the susceptible strain regardless of VPA status. Based on this information, we hypothesized that maternal supplementation with L-carnitine (400 mg/kg), coenzyme A (200 mg/kg), or hypotaurine (350 mg/kg) would reduce VPA-induced NTDs in the sensitive strain and found that administration of hypotaurine prior to VPA exposure significantly reduced the occurrence of NTDs by close to one-third compared to controls. L-carnitine and coenzyme A reduced resorption rates but did not significantly reduce NTD risk in the sensitive strain. These results suggest that genetic variants or environmental exposures influencing embryonic hypotaurine status may be factors in determining risk for adverse pregnancy outcomes when managing the health care needs of pregnant women exposed to VPA or other ASMs.

Genetic basis of susceptibility to teratogen induced birth defects.

Birth defects remain the leading cause of infant death in US. The field of teratology has been focused on the causes and underlying mechanisms of birth defects for decades, yet our understanding of these critical issues remain unacceptably vague. Conclusions from years of animal and human studies made it clear that the vast majority of birth defects have multifactorial origins, with contributions from environmental and genetic factors. The environment comprises not only of the physical, biological, and chemical external environment surrounding the pregnant woman, but it also includes the internal environment of the woman's body that interact with the developing embryo in a complex fashion. The importance of maternal and embryonic genetic factors consisting of countless genetic variants/mutations that exist within every individual contribute to birth defect susceptibility is only now being more fully appreciated. This great complexity of the genome and its diversity within individuals and populations seems to be the principal reason why the same teratogenic exposure can induce severe malformation in one embryo, while fail to do so to other exposed embryos. As the interaction between genetic and environmental factors has long been recognized as the first "Principle of Teratology" by Wilson and Warkany [1965. Teratology: Principles and techniques. Chicago: University of Chicago Press], it is only recently that the appropriate investigative tools have been developed with which to fully investigate this fundamental principle. The introduction of high throughput technologies like whole genome sequencing or genome-wide association studies are promising to deliver an enormous amount of new data that will shed light on the genomic factors that contribute susceptibility to environmental teratogens. In this review, we attempt to summarize the epidemiological and experimental literature concerning birth defects whose phenotypic expression can be clearly related to the interactions between several select environmental factors and those genetic pathways in which they are most likely to have significant modifying effects. © 2011 Wiley-Liss, Inc.