Differential response of heterozygous curly-tail mouse embryos to vitamin A teratogenesis depending on maternal genotype

Around 60% of mice homozygous for the curly-tail gene have neural tube defects (NTD). F1 hybrids between curly-tail mice (ct) and a nongenetically predisposed A strain (A) do not have NTD. Vitamin A increases the penetrance of the ct gene at doses that hardly cause any NTD in A mice. The susceptibility to vitamin A of the F1 hybrids, derived from ct female X A male and A female X ct male crosses, was examined. Either 10, 20, or 40 mg/kg was injected into the mother on the eighth day of pregnancy. The F1 hybrids did develop NTD, and there was a dose response. The number with NTD was roughly intermediate between that found in the two pure parental crosses. However, there was a significant difference in response according to whether the ct or the A was the mother, more F1 embryos having NTD in the former situation. Also, significantly more of the female F1 hybrids had NTD when the ct was the mother than when the A was the mother, but for the males, the number with NTD was the same in both crosses. The number of intrauterine deaths of F1 hybrids, seen as resorptions in each reciprocal cross, was not intermediate but matched that of the pure parental strain of the mother involved in the hybrid cross. It is concluded that gene-environment interaction has been demonstrated, together with both a maternal effect and a contribution of the fetal genotype in the liability to produce NTD in response to vitamin A.     

Neural tube defects in curly-tail mice. I. Incidence, expression and similarity to the human condition.

The incidence of neurovertebral defects in mutant mice of the curly-tail strain was investigated and found to be similar to that observed in the same mice twenty-five years ago. The results of breeding experiments support the hypothesis of Grüneberg that the defects in these mice are probably caused by a recessive gene, the expression of which is markedly affected by the genetic background. Selection against the curly-tail phenotype for six generations did not affect the incidence of abnormalities. A marked excess of females was found among exencephalic mice, as among humans with neural tube defects. Similarly, polyhydramnios, hydrocephaly, high levels of amniotic fluid alphafoetoprotein and distinctive, rapidly adhering cells in the amniotic fluid also occurred in these mice, as in humans. The curly-tail mice thus provide a useful model for the investigation of neural tube defects in man.     

Hyperthermia and neural tube defects of the curly-tail mouse

The mutant gene curly-tail produces neural tube defects (NTD) in 60% of mice, predominantly at the caudal end of the neural tube. Only 1% of individuals have exencephaly. Pregnant curly-tail mice and C57BL mice which are not genetically pre-disposed to NTD, were subjected to various regimes of hyperthermia on day 8 or on day 9 or on day 10 of gestation. Normal body temperature was around 36.8 degrees C, but it was found to be extremely labile in response to heat exposure. It was significantly raised for 15 min of a 20-min exposure period, and, after removal from the heat, it dropped rapidly. In C57BL mice, heat treatment produced exencephaly alone and in only 3% of mice. In curly-tail mice, none of the heat-treatment regimes had any consistent effect on the incidence of posterior NTD but produced specifically exencephaly. The incidence was increased slightly at an environmental temperature of 37 degrees C when the body temperature was 4.01 degrees C; at an ambient temperature of 43 degrees C and a body temperature of 42 degrees C, the incidence of exencephaly was 20%. Exencephaly was produced by two periods of 20 min heat exposures 7 hr apart or a single exposure of 1 hr, especially on day 8 of gestation, but not by a single 20 min exposure. It is concluded that these experiments, performed in a mutant predisposed to lesions especially at the caudal end of the neural tube, demonstrate the specificity of hyperthermia for affecting closure of the cranial neural folds.     

Effect of mitomycin C on the neural tube defects of the curly-tail mouse

Around 60% of the mouse mutants called curly-tail, have tail aberrations in the form of a coil or a kink, with or without lumbosacral spina bifida, and rarely, exencephaly. These neural tube defects (NTD) are the result of an incompletely penetrant recessive gene. A single injection of various doses (1-6 mg/kg) of the DNA inhibitor mitomycin C was given to pregnant curly-tail mice on day 7, 8, or 9 of gestation, and its effect on the NTD of the embryos was noted. No dose used was lethal to the embryo. When given on day 7 or day 8, mitomycin C markedly increased the number of exencephalics, and additionally, on day 8, it reduced the number of posterior abnormalities. However, on day 9, no exencephaly was produced, and there was a drastic reduction in the number of tail and spinal defects, the overall incidence of NTD being as low as 15% with 2 mg/kg. A twofold effect of mitomycin C on the curly-tail embryos was thus observed--according to the time in development it was administered, firstly, a teratogenic effect, and later, a "remedial" or preventive effect.     

Effect of hydroxyurea on neural tube defects in the curly-tail mouse

Around 60% of curly-tail mice spontaneously develop neural tube defects (NTD), that is, exencephaly, and/or spina bifida (open lesions), or a curly tail (closed lesion), due to an incompletely penetrant recessive gene. Various doses of hydroxyurea, a teratogen to the rodent central nervous system, were administered to curly-tail mice on either day 8 or day 9 of pregnancy in an attempt to increase the number of NTD in the embryos. No dose used on either day achieved this. However, on day 8, the proportion of affected mice with open lesions increased from around 30% in control mice to 78% with 400 mg/kg hydroxyurea, and this was accounted for specifically by the production of exencephaly. When administered on day 9, 400, 500, and 600 mg/kg hydroxyurea (the latter two doses being lethal to embryos on day 8) actually reduced the incidence of total NTD, to around 30%. Among these affected mice, even though reduced in number, there was still a slight tendency for an increase in the number of exencephalics. Hydroxyurea also produced gastroschisis in a small percentage of embryos; the greatest incidence was 36% with 400 mg/kg on day 8.     

Methylenetetrahydrofolate reductase and transcobalamin genetic polymorphisms in human spontaneous abortion: biological and clinical implications

The pathogenesis of human spontaneous abortion involves a complex interaction of several genetic and environmental factors. The firm association between increased homocysteine concentration and neural tube defects (NTD) has led to the hypothesis that high concentrations of homocysteine might be embryotoxic and lead to decreased fetal viability. There are several genetic polymorphisms that are associated with defects in folate- and vitamin B₁₂-dependent homocysteine metabolism. The methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C polymorphisms cause elevated homocysteine concentration and are associated with an increased risk of NTD. Additionally, low concentration of vitamin B₁₂ (cobalamin) or transcobalamin that delivers vitamin B₁₂ to the cells of the body leads to hyperhomocysteinemia and is associated with NTD. This effect involves the transcobalamin (TC) 776C>G polymorphism. Importantly, the biochemical consequences of these polymorphisms can be modified by folate and vitamin B₁₂ supplementation. In this review, I focus on recent studies on the role of hyperhomocysteinemia-associated polymorphisms in the pathogenesis of human spontaneous abortion and discuss the possibility that periconceptional supplementation with folate and vitamin B₁₂ might lower the incidence of miscarriage in women planning a pregnancy.     

Expression of a new mutation (Axd) causing axial defects in mice correlates with maternal phenotype and age

A new autosomal mutation, Axd (axial defects), is described. Axd segregates in a simple Mendelian fashion, and it is dominant with incomplete penetrance and variable expressivity. The phenotype of Axd heterozygotes ranges from a variety of tail anomalies to visibly normal tails. Approximately 12% of neonates from curly-tail (CT) F1 (Axd/+) x F1 (Axd/+) matings exhibit open neural tube defects (NTD) in the lumbosacral region and 16% have curly tails. Mean litter sizes and resorption rates comparable to wild type indicate that homozygosity for Axd is not obligately lethal. Genetic background plays a major role in Axd expression. Strains such as BALB/cByJ allow the highest penetrance of the mutation in single dose (46%), whereas, in CF-1 mice Axd is recessive. The tail phenotype of heterozygous Axd/+ dams, in part reflective of their genetic background, correlates with the incidence of NTD in F2 offspring: CT mothers produce significantly more neonates with frank NTD than normal tail mothers. At the one embryonic period examined for this study (D13/D14 post-coitus), an 85% higher incidence of total axial defects is observed than among the F2 at birth. Unchanging litter size and the relative increase in phenotypically normal offspring by birth suggest that Axd acts by delaying posterior neural tube closure. One of the most significant findings in this study is that maternal age influences the survival of Axd embryos in utero. Axd/+ dams older than 8 months yield fewer mean implants, higher resorption rates, and fewer viable embryos with axial defects than do Axd/+ dams younger than 8 months. Axd is not allelic to nor linked to the Sp (splotch) gene which also affects neurulation.     

Genetic basis of neural tube defects. I. Regulatory genes for the neurulation process

Neural tube defects (NTD) together with cardiovascular system defects are the most common malformations in the Polish population (2.05-2.68/1000 newborns). They arise during early embryogenesis and are caused by an improper neural groove closure during the neurulation process. NTD can arise from the influence of specific environmental factors on the foetus. The genetic factor is also very important, because NTDs have multigenetic conditioning. It was suggested that genes connected with the regulation of neurulation could also be involved in NTD aetiology, especially when their deletion or modification leads to neural tube defects in the mouse model. Examples are genes from the PAX family, T (Brachyury), BRCA1 and PDGFRA genes.     

Impact of methylenetetrahydrofolate reductase deficiency and low dietary folate on the development of neural tube defects in splotch mice

BACKGROUND: The etiology of neural tube defects (NTDs) is multifactorial, with environmental and genetic determinants. Folate supplementation prevents the majority of NTDs, and a polymorphism in methylenetetrahydrofolate reductase (MTHFR) has become recognized as a genetic risk factor. The mechanisms by which folate affects NTD development are unclear. The Splotch (Sp) mouse is a well-characterized mouse model for studying spontaneous NTDs. To assess the potential interaction between folate metabolism and the Sp mutant in NTD development, we studied mice with both Sp and Mthfr mutations, as well as the interaction between Sp and low dietary folate. METHODS: Wild-type, single Mthfr+/-mutant, single Sp/+mutant, and double mutant (Mthfr+/-, Sp/+) female mice were mated with males of the same genotype. Embryos were examined for NTDs on gestational day (GD) 13.5. To investigate the effects of folate deficiency on Sp mice, Sp/+female mice were fed a control diet (CD), a moderately folic acid-deficient diet (MFADD), or a severely folic acid-deficient diet (SFADD). They were mated with Sp/+males and the embryos were examined. RESULTS: There were no differences in the incidence or severity of NTDs in embryos from double-mutant mating pairs compared to those from single Sp mutants. Embryos from Mthfr+/-dams did not exhibit NTDs. Diets deficient in folate did not influence the incidence or severity of NTDs in embryos from Sp/+mice. CONCLUSIONS: We did not observe an interaction between Sp and Mthfr mutations, or between the Sp mutation and low dietary folate, in NTD development in Splotch mice.     

Expression of the Axd (axial defects) mutation in the mouse is insensitive to retinoic acid at low dose

The Axd mutation in the mouse acts by an unknown mechanism to cause lumbosacral open neural tube defects and a variety of tail anomalies. Retinoic acid (RA) plays a number of different physiological and developmental roles and has been shown to affect neurulation in mice and other species. Indeed, reports have shown that this biologically active compound (or its metabolites) at low dose can alter the incidence of neural tube defects (NTD) in curly-tail (ct), splotch (Sp), and delayed splotch (Spd) mice, strains that are genetically predisposed to such abnormalities. The aim of the present study was to determine if RA administered under similar conditions would affect the penetrance or expression of the Axd mutation or survival of Axd homozygotes. Axd/+ and +/+ dams were exposed to RA intraperitoneally (5 mg/kg) on D9 postcoitus. No difference in incidence or extent of neural tube defects or other axial anomalies was detected among embryos of Axd/+ dams given RA compared with those administered vehicle only. This finding is consistent with the diversity of gene-controlled steps required for neurulation and the differing sensitivities of specific mutants to rescue by extrinsic agents.     

<Emphasis Type="Italic">Pax1/E2a</Emphasis> Double-Mutant Mice Develop Non-Lethal Neural Tube Defects that Resemble Human Malformations

Many mouse models exist for neural tube defects (NTDs), but only few of them are relevant for human patients that are born alive with spina bifida aperta. NTDs in humans show a complex inheritance, which most likely result from the involvement of a variety of predisposing genetic and environmental factors. Hints toward the identity of predisposing genetic factors for human NTDs could come from mouse studies on the development of the neural tube and spinal cord, as well as from studies on associated features of this type of diseases. Among such features is the observation that pregnancies affected by a neural tube defect frequently show changes in thymus morphology, and in both neonatal and maternal T-cell repertoire. The genes for E2a and Pax1 have both been implicated in not only paraxial mesodermal development, but also in that of the immune system. Moreover, Pax1 mutant mice have been shown to display NTDs in digenic mouse models. In the present study we have investigated the phenotype of E2a null mutant mice that are also heterozygous for the so-called undulated mutation in Pax1. Here we report that such double-mutant mice develop a non-lethal NTD that strongly resembles the classic human NTD: spina bifida aperta, associated with defects of the axial skeleton, immune system and urinary tract.     

Folate-mediated one-carbon metabolism and neural tube defects: balancing genome synthesis and gene expression

Neural tube defects (NTDs) refer to a cluster of neurodevelopmental conditions associated with failure of neural tube closure during embryonic development. Worldwide prevalence of NTDs ranges from approximately 0.5 to 60 per 10,000 births, with regional and population-specific variation in prevalence. Numerous environmental and genetic influences contribute to NTD etiology; accumulating evidence from population-based studies has demonstrated that folate status is a significant determinant of NTD risk. Folate-mediated one-carbon metabolism (OCM) is essential for de novo nucleotide biosynthesis, methionine biosynthesis, and cellular methylation reactions. Periconceptional maternal supplementation with folic acid can prevent occurrence of NTDs in the general population by up to 70%; currently several countries fortify their food supply with folic acid for the prevention of NTDs. Despite the unambiguous impact of folate status on NTD risk, the mechanism by which folic acid protects against NTDs remains unknown. Identification of the mechanism by which folate status affects neural tube closure will assist in developing more efficacious and better targeted preventative measures. In this review, we summarize current research on the relationship between folate status and NTDs, with an emphasis on linking genetic variation, folate nutriture, and specific metabolic and/or genomic pathways that intersect to determine NTD outcomes.     

Metabolic effects and the methylenetetrahydrofolate reductase (MTHFR) polymorphism associated with neural tube defects in southern Brazil

BACKGROUND: The importance of metabolic factors in neural tube defects (NTDs) has been the focus of many investigations. Several authors have suggested that abnormalities in homocysteine metabolism, such as hyperhomocysteinemia, folate deficiency, and low vitamin B12, may be responsible for these malformations and that both nutritional factors and genetic abnormalities are associated with them. METHODS: We conducted a case-control study to investigate the influence of biochemical and genetic factors in NTDs in infants in southern Brazil. Levels of folate, vitamin B12, total homocysteine (t-Hcy) and the 677C>T and 1298A>C polymorphisms of the MTHFR gene were analyzed in 41 NTD child-mother pairs and 44 normal child-mother control pairs. RESULTS: Subjects in the case group had a higher mean blood folate level than those in the control group. The level of vitamin B12 was lower in mothers in the NTD group than in control mothers (p = 0.004). The level of t-Hcy was not different in the two groups, but t-Hcy and vitamin B12 were correlated (p = 0.002). There was no difference in the genotype distribution for 677C>T and 1298A>C polymorphisms of MTHFR in the case and control pairs. The level of t-Hcy was correlated with 677TT. CONCLUSIONS: Despite the small sample in this study, we suggest that low vitamin B12 and, consequently, hyperhomocysteinemia are important risk factors for NTDs in our population.     

同型半胱氨酸与神经管畸形的相关病因学研究进展

神经管畸形(neural tube defects,NTDs)是一种最常见的严重中枢神经系统先天性畸形,在世界各地均有发生。它是造成流产、死产的主要原因之一,即便胎儿存活,也严重影响患儿的生长发育和生活质量,同时给家庭和社会带来沉重的精神压力和经济负担。神经管畸形的发生绝大多数是由遗传因素与环境因素相互作用的结果,若孕妇在孕早期缺乏叶酸、高热、接触射线、服用药物、感染或发生妊娠期糖尿病等条件下,结合遗传因素作用,均有可能导致神经管畸形的发生,但其目前其的确切病因及其发病机制仍有待深入研究。大量研究表明,在孕妇血清中低叶酸、低维生素B12水平及高血浆同型半胱氨酸(Hcy)水平,都与NTDs的发生密切相关。本文主要围绕同型半胱氨酸的代谢,从分子水平和基因水平对与神经管畸形相关的各因素做一综述。     

Neural tube defects and folate: case far from closed

Neural tube closure takes place during early embryogenesis and requires interactions between genetic and environmental factors. Failure of neural tube closure is a common congenital malformation that results in morbidity and mortality. A major clinical achievement has been the use of periconceptional folic acid supplements, which prevents approximately 50-75% of cases of neural tube defects. However, the mechanism underlying the beneficial effects of folic acid is far from clear. Biochemical, genetic and epidemiological observations have led to the development of the methylation hypothesis, which suggests that folic acid prevents neural tube defects by stimulating cellular methylation reactions. Exploring the methylation hypothesis could direct us towards additional strategies to prevent neural tube defects.     

A unique missense allele of BAF155, a core BAF chromatin remodeling complex protein,causes neural tube closure defects in mice

Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155^msp3, a unique ENU‐induced allele in mice. Homozygous Baf155^mps3 embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA‐Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 483–497, 2014     

A screen for mutations in human homologues of mice exencephaly genes Tfap2alpha and Msx2 in patients with neural tube defects

BACKGROUND: Very little is known about the identity of genetic factors involved in the complex etiology of nonsyndromic neural tube defects (NTD). Potential susceptibility genes have emerged from the vast number of mutant mouse strains displaying NTD. Reasonable candidates are the human homologues of mice exencephaly genes Tfap2alpha and Msx2, which are expressed in the developing neural tube. METHODS: A single-strand conformation analysis (SSCA) mutation screen of the coding sequences of TFAP2alpha and MSX2 was performed for 204 nonsyndromic NTD patients including cases of anencephaly (n = 10), encephalocele (n = 8), and spina bifida aperta, SBA (n = 183). A selected number of SBA patients was additionally tested for specific mutations in MTHFD, FRalpha, and PAX1 already shown to be related to NTD. RESULTS: Two TFAP2alpha point mutations in individual SBA patients were silent on the amino acid level (C308C, T396T). On nucleic acid level, these mutations change evolutionary conserved codons and thus may influence mRNA processing and translation efficiency. One SBA patient displayed an exonic 9-bp deletion in MSX2 leading to a shortened and possibly less functional protein. None of these mutations was found in 222 controls. Seven polymorphisms detected in TFAP2alpha and MSX2 were equally distributed in patients and controls. Patients with combined heterozygosity of an exonic MSX2 and an intronic TFAP2alpha polymorphism were at a slightly increased risk of NTD (OR 1.71; 95% CI 0.57-5.39). CONCLUSIONS: Although several new genetic variants were found in TFAP2 and MSX2, no statistically significant association was found between NTD cases and the new alleles or their combinations. Further studies are necessary to finally decide if these gene variants may have acted as susceptibility factors in our individual cases.     

Mouse as a model for multifactorial inheritance of neural tube defects

Neural tube defects (NTDs) such as spina bifida and anencephaly are some of the most common structural birth defects found in humans. These defects occur due to failures of neurulation, a process where the flat neural plate rolls into a tube. In spite of their prevalence, the causes of NTDs are poorly understood. The multifactorial threshold model best describes the pattern of inheritance of NTDs where multiple undefined gene variants interact with environmental factors to cause an NTD. To date, mouse models have implicated a multitude of genes as required for neurulation, providing a mechanistic understanding of the cellular and molecular pathways that control neurulation. However, the majority of these mouse models exhibit NTDs with a Mendelian pattern of inheritance. Still, many examples of multifactorial inheritance have been demonstrated in mouse models of NTDs. These include null and hypomorphic alleles of neurulation genes that interact in a complex fashion with other genetic mutations or environmental factors to cause NTDs. These models have implicated several genes and pathways for testing as candidates for the genetic basis of NTDs in humans, resulting in identification of putative pathogenic mutations in some patients. Mouse models also provide an experimental paradigm to gain a mechanistic understanding of the environmental factors that influence NTD occurrence, such as folic acid and maternal diabetes, and have led to the discovery of additional preventative nutritional supplements such as inositol. This review provides examples of how multifactorial inheritance of NTDs can be modeled in the mouse. Birth Defects Research (Part C) 96:193–205, 2012. © 2012 Wiley Periodicals, Inc.