Over-expression of Grhl2 causes spina bifida in the Axial defects mutant mouse

Cranial neural tube defects (NTDs) occur in mice carrying mutant alleles of many different genes, whereas isolated spinal NTDs (spina bifida) occur in fewer models, despite being common human birth defects. Spina bifida occurs at high frequency in the Axial defects (Axd) mouse mutant but the causative gene is not known. In the current study, the Axd mutation was mapped by linkage analysis. Within the critical genomic region, sequencing did not reveal a coding mutation whereas expression analysis demonstrated significant up-regulation of grainyhead-like 2 (Grhl2) in Axd mutant embryos. Expression of other candidate genes did not differ between genotypes. In order to test the hypothesis that over-expression of Grhl2 causes Axd NTDs, we performed a genetic cross to reduce Grhl2 function in Axd heterozygotes. Grhl2 loss of function mutant mice were generated and displayed both cranial and spinal NTDs. Compound heterozygotes carrying both loss (Grhl2 null) and putative gain of function (Axd) alleles exhibited normalization of spinal neural tube closure compared with Axd/+ littermates, which exhibit delayed closure. Grhl2 is expressed in the surface ectoderm and hindgut endoderm in the spinal region, overlapping with grainyhead-like 3 (Grhl3). Axd mutants display delayed eyelid closure, as reported in Grhl3 null embryos. Moreover, Axd mutant embryos exhibited increased ventral curvature of the spinal region and reduced proliferation in the hindgut, reminiscent of curly tail embryos, which carry a hypomorphic allele of Grhl3. Overall, our data suggest that defects in Axd mutant embryos result from over-expression of Grhl2.     

D-chiro-inositol is more effective than myo-inositol in preventing folate-resistant mouse neural tube defects

BACKGROUND: Among mouse genetic mutants that develop neural tube defects (NTDs), some respond to folic acid administration during early pregnancy, whereas NTDs in other mutants are not prevented. This parallels human NTDs, in which up to 30% of cases may be resistant to folic acid. Most spina bifida cases in the folic acid-resistant 'curly tail' mouse can be prevented by treatment with inositol early in embryonic development. Here, the effectiveness and safety during pregnancy of two isomers, myo- and D-chiro-inositol, in preventing mouse NTDs was compared. METHODS AND RESULTS: Inositol was administered either directly to embryos in vitro, or to pregnant females by s.c. or oral routes. Although D-chiro- and myo-inositol both reduced the frequency of spina bifida in curly tail mice by all routes of administration, D-chiro-inositol consistently exhibited the more potent effect, reducing spina bifida by 73-86% in utero compared with a 53-56% reduction with myo-inositol. Pathological analysis revealed no association of either myo- or D-chiro-inositol with reduced litter size or fetal malformation. CONCLUSIONS: D-chiro-inositol offers a safe and effective method for preventing folic acid-resistant NTDs in the curly tail mouse. This raises the possibility of using inositol as an adjunct therapy to folic acid for prevention of NTDs in humans.     

Does lumbosacral spina bifida arise by failure of neural folding or by defective canalisation?

The aim of this study was to determine whether open lumbosacral spina bifida results from an abnormality of neural folding (primary neurulation) or medullary cord canalisation (secondary neurulation). Homozygous curly tail (ct) mouse embryos were studied as a model system for human neural tube defects. The rostral end of the spina bifida was found to lie at the level of somites 27 to 32 in over 90% of affected ct/ct embryos. Indian ink marking experiments using non-mutant embryos showed that the posterior neuropore closes, and primary neurulation is completed, at the level of somites 32 to 34. Since neurulation in mammals progresses in a craniocaudal sequence, without overlap between regions of primary and secondary neurulation, we conclude that spina bifida in ct/ct embryos arises initially as a defect of primary neurulation. The position of posterior neuropore closure in human embryos is estimated to lie at the level of the future second sacral segment indicating that in humans, as in the ct mouse, lumbosacral spina bifida usually arises as a defect of posterior neuropore closure. Cranial NTD affect females predominantly, whereas lower spinal NTD are more common in males, both in humans and ct mice. We offer an explanation for this phenomenon based on (a) differences in the effect of embryonic growth retardation on the likelihood that an embryo will develop either cranial or lower spinal NTD and (b) differences in the rate of growth and development of male and female embryos at the time of neurulation.     

Curly tail: a 50-year history of the mouse spina bifida model

This paper reviews 50 years of progress towards understanding the aetiology and pathogenesis of neural tube defects (NTD) in the curly tail (ct) mutant mouse. More than 45 papers have been published on various aspects of curly tail with the result that it is now the best understood mouse model of NTD pathogenesis. The failure of closure of the spinal neural tube, which leads to spina bifida in this mouse, has been traced back to a tissue-specific defect of cell proliferation in the tail bud of the E9.5 embryo. This cell proliferation defect results in a growth imbalance in the caudal region that generates ventral curvature of the body axis. Neurulation movements are opposed, leading to delayed neuropore closure and spina bifida, or tail defects. It is interesting to reflect that these advances have been achieved in the absence of information on the nature of the ct gene product, which remains unidentified. In addition to the principal ct gene, which maps to distal Chromosome 4, the curly tail phenotype is influenced by several modifier genes and by environmental factors. NTD in curly tail are resistant to folic acid, as is thought to be the case in 30% of human NTD, whereas they can be prevented by myo-inositol. These and other features of NTD in this system bear striking similarities to the situation in humans, making curly tail a model for understanding a sub-type folic acid-resistant human NTD.     

Mouse Fkbp8 activity is required to inhibit cell death and establish dorso-ventral patterning in the posterior neural tube

Neural tube defects (NTDs) are birth defects that can be disabling or lethal and are second in their prevalence after cardiac defects among major human congenital malformations. Spina bifida is a NTD where the spinal cord is dysplastic, and the overlying spinal column is absent. At present, the molecular mechanisms underlying the spinal bifida development are largely unknown. In this study, we present a Fkbp8 mouse mutant that has an isolated and completely penetrant spina bifida, which is folate- and inositol-resistant. Fkbp8 mutants are not embryo lethal, but they display striking features of human spina bifida, including a dysplastic spinal cord, open neural canal and disability. The loss of Fkbp8 leads to increased apoptosis in the posterior neural tube, demonstrating that in vivo FKBP8 inhibits cell death. Gene expression analysis of Fkbp8 mutants revealed a perturbation of expression of neural tube patterning genes, suggesting that endogenous FKBP8 activity establishes dorso-ventral patterning of the neural tube. These studies demonstrate that Fkbp8 is not important for embryo survival, but is essential for spinal neural tube patterning, and to block apoptosis, in the developing neural tube. The mutant Fkbp8 allele is a new experimental model which will be useful in dissecting the pathogenesis of spinal NTDs, and enhance our understanding of the etiology of human NTDs.     

Contribution of VANGL2 mutations to isolated neural tube defects

Kibar Z, Salem S, Bosoi CM, Pauwels E, De Marco P, Merello E, Bassuk AG, Capra V, Gros P. Contribution of VANGL2 mutations to isolated neural tube defects. Vangl2 was identified as the gene defective in the Looptail (Lp) mouse model for neural tube defects (NTDs). This gene forms part of the planar cell polarity (PCP) pathway, also called the non‐canonical Frizzled/Dishevelled pathway, which mediates the morphogenetic process of convergent extension essential for proper gastrulation and neural tube formation in vertebrates. Genetic defects in PCP signaling have strongly been associated with NTDs in mouse models. To assess the role of VANGL2 in the complex etiology of NTDs in humans, we resequenced this gene in a large multi‐ethnic cohort of 673 familial and sporadic NTD patients, including 453 open spina bifida and 202 closed spinal NTD cases. Six novel rare missense mutations were identified in seven patients, five of which were affected with closed spinal NTDs. This suggests that VANGL2 mutations may predispose to NTDs in approximately 2.5% of closed spinal NTDs (5 in 202), at a frequency that is significantly different from that of 0.4% (2 in 453) detected in open spina bifida patients (p = 0.027). Our findings strongly implicate VANGL2 in the genetic causation of spinal NTDs in a subset of patients and provide additional evidence for a pathogenic role of PCP signaling in these malformations.     

Specific isoforms of protein kinase C are essential for prevention of folate-resistant neural tube defects by inositol

A proportion of neural tube defects (NTDs) can be prevented by maternal folic acid supplementation, although some cases are unresponsive. The curly tail mutant mouse provides a model of folate-resistant NTDs, in which defects can be prevented by inositol therapy in early pregnancy. Hence, inositol represents a possible novel adjunct therapy to prevent human NTDs. The present study investigated the molecular mechanism by which inositol prevents mouse NTDs. Activation of protein kinase C (PKC) is known to be essential, and we examined neurulation-stage embryos for PKC expression and applied PKC inhibitors to curly tail embryos developing in culture. Although all known PKC isoforms were detected in the closing neural tube, use of chemical PKC inhibitors identified a particular requirement for 'conventional' PKC isoforms. Peptide inhibitors offer selective inhibition of individual PKCs, and we demonstrated isoform-specific inhibition of PKC in embryonic cell cultures. Application of peptide inhibitors to neurulation-stage embryos revealed an absolute dependence on the activity of PKCbetaI and gamma for prevention of NTDs by inositol, and partial dependence on PKCzeta, whereas other PKCs (alpha, betaII delta, and epsilon) were dispensable. To investigate the cellular action of inositol and PKCs in NTD prevention, we examined cell proliferation in curly tail embryos. Defective proliferation of hindgut cells is a key component of the pathogenic sequence leading to NTDs in curly tail. Hindgut cell proliferation was stimulated specifically by inositol, an effect that required activation of PKCbetaI. Our findings reveal an essential role of specific PKC isoforms in mediating the prevention of mouse NTDs by inositol.     

Folic acid supplementation can adversely affect murine neural tube closure and embryonic survival

Neural tube defects (NTDs), a common birth defect in humans, result from the failure of the embryonic neural tube (NT) to close properly. NT closure is a complex, poorly understood morphogenetic process influenced by genes and environment. The most effective environmental influence in decreasing the risk for NTDs is folic acid (FA) fortification and supplementation, and these findings led to the recommendation of periconceptual FA intake and mandatory fortification of the US grain supply in 1998. To explore the relationship between genetics and responsiveness to FA supplementation, we used five mouse NTDs models-Zic2, Shroom3, Frem2, Grhl2 (Grainyhead-like 2) and L3P (Line3P)-and a long-term generational FA supplementation scheme. Contrary to expectations, we find that three genetic mutants respond adversely to FA supplementation with increased incidence of NTDs in homozygous mutants, occurrence of NTDs in heterozygous embryos and embryonic lethality prior to NT closure. Because of these unexpected responses, we examined NTD risk after short-term FA supplementation. Our results indicate that, for the same genetic allele, NTD risk can depend on the length of FA exposure. Our data indicate that, depending on the gene mutation, FA supplementation may adversely influence embryonic development and NT closure.     

A known functional polymorphism (Ile120Val) of the human PCMT1 gene and risk of spina bifida

Folate binding protein 1 (Folr1) knockout mice with low maternal folate concentrations have been shown to be excellent animal models for human folate-responsive neural tube defects (NTDs). Previous studies using the Folr1 knockout mice revealed that maternal folate supplementation up-regulates the expression of the PCMT1 gene in Folr1 nullizygous neural tube tissue during neural tube closure. PCMT1 encodes the protein repair enzyme l-isoaspartate (d-aspartate) O-methyltransferase (PIMT) that converts abnormal d-aspartyl and l-isoaspartyl residues to the normal l-aspartyl form. PIMT is known to protect certain neural cells from Bax-induced apoptosis. Pcmt1-deficient mice present with abnormal AdoMet/AdoHcy homeostasis. We hypothesized that a known functional polymorphism (Ile120Val) in the human PCMT1 gene is associated with an increased risk of folate-responsive human NTDs. A case-control study was conducted to investigate a possible association between this polymorphism and risk of spina bifida. Compared to the Ile/Ile and Ile/Val genotypes, the homozygous Val/Val genotype showed decreased risk for spina bifida (adjusted odds ratio=0.6, 95% confidence interval: 0.4-0.9). Our results showed that the Ile120Val polymorphism of PCMT1 gene is a genetic modifier for the risk of spina bifida. Val/Val genotype was associated with a reduction in risk for spina bifida.     

Mutations in the planar cell polarity gene, Fuzzy, are associated with neural tube defects in humans

Neural tube defects (NTDs) are a heterogeneous group of common severe congenital anomalies which affect 1-2 infants per 1000 births. Most genetic and/or environmental factors that contribute to the pathogenesis of human NTDs are unknown. Recently, however, pathogenic mutations of VANGL1 and VANGL2 genes have been associated with some cases of human NTDs. Vangl genes encode proteins of the planar cell polarity (PCP) pathway that regulates cell behavior during early stages of neural tube formation. Homozygous disruption of PCP genes in mice results in a spectrum of NTDs, including defects that affect the entire neural axis (craniorachischisis), cranial NTDs (exencephaly) and spina bifida. In this paper, we report the dynamic expression of another PCP gene, Fuzzy, during neural tube formation in mice. We also identify non-synonymous Fuzzy amino acid substitutions in some patients with NTDs and demonstrate that several of these Fuzzy mutations affect formation of primary cilia and ciliary length or affect directional cell movement. Since Fuzzy knockout mice exhibit both NTDs and defective primary cilia and Fuzzy is expressed in the emerging neural tube, we propose that mutations in Fuzzy may account for a subset of NTDs in humans.     

Mouse models of neural tube defects: investigating preventive mechanisms

Neural tube defects (NTD), including anencephaly and spina bifida, are a group of severe congenital abnormalities in which the future brain and/or spinal cord fail to close. In mice, NTD may result from genetic mutations or knockouts, or from exposure to teratogenic agents, several of which are known risk factors in humans. Among the many mouse NTD models that have been identified to date, a number have been tested for possible primary prevention of NTD by exogenous agents, such as folic acid. In genetic NTD models such as Cart1, splotch, Cited2, and crooked tail, and NTD induced by teratogens including valproic acid and fumonisins, the incidence of defects is reduced by maternal folic acid supplementation. These folate-responsive models provide an opportunity to investigate the possible mechanisms underlying prevention of NTD by folic acid in humans. In another group of mouse models, that includes curly tail, axial defects, and the Ephrin-A5 knockout, NTD are not preventable by folic acid, reflecting the situation in humans in which a subset of NTD appear resistant to folic acid therapy. In this group of mutants alternative preventive agents, including inositol and methionine, have been shown to be effective. Overall, the data from mouse models suggests that a broad-based in utero therapy may offer scope for prevention of a greater proportion of NTD than is currently possible.     

A genetic risk factor for mouse neural tube defects: defining the embryonic basis

Genetic polymorphisms are thought to play an important role in determining susceptibility to neural tube defects (NTDs), for example between different ethnic groups, but the embryonic manifestation of these polymorphic genetic influences is unclear. We have used a mouse model to test experimentally whether polymorphic variations in the pattern of cranial neural tube closure can influence susceptibility to NTDs. The site at which cranial neural tube closure begins (so-called closure 2) is polymorphic between inbred mice. Strains with a caudal location of closure 2 (e.g. DBA/2) are relatively resistant to NTDs, whereas strains with a rostrally positioned closure 2 (e.g. NZW) exhibit increased susceptibility to NTDs. We tested experimentally whether altering the position of closure 2 can affect susceptibility to cranial NTDs, by back- crossing the splotch ( Sp (2H) ) mutant gene onto the DBA/2 background. As a control, Sp (2H) was transferred onto the NZW background, which resembles splotch mice in its closure pattern. Approximately 80% of Sp (2H) homozygotes develop NTDs, both cranial (exencephaly) and spinal (spina bifida). After transfer to the DBA/2 background, the frequency of cranial NTDs was reduced significantly in Sp (2H) homozygotes, confirming a protective effect of caudal closure 2. In contrast, Sp (2H) homozygotes on the NZW background had a persistently high frequency of cranial NTDs. The frequency of spina bifida was not altered in either backcross, emphasizing the specificity of this genetic effect for cranial neurulation. These findings demonstrate that variation in the pattern of cranial neural tube closure is a genetically determined factor influencing susceptibility to cranial NTDs.     

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.     

Testing for genetic associations in a spina bifida population: analysis of the HOX gene family and human candidate gene regions implicated by mouse models of neural tube defects

Neural tube defects (NTDs) are among the most common severely disabling birth defects in the United States, affecting approximately 1-2 of every 1,000 live births. The etiology of NTDs is multifactorial, involving the combined action of both genetic and environmental factors. HOX genes play a central role in establishing the initial body plan by providing positional information along the anterior-posterior body and limb axis and have been implicated in neural tube closure. There are many mouse models that exhibit both naturally occurring NTDs in various mouse strains as well as NTDs that have been created by "knocking out" various genes. A nonparametric linkage method, the transmission disequilibrium test (TDT), was utilized to test the HOX gene family and human equivalents of genes (when known) or the syntenic region in humans to those in mouse models which could play a role in the formation of NTDs. DNA from 459 spina bifida (SB) affected individuals and their parents was tested for linkage and association utilizing polymorphic markers from within or very close to the HOXA, HOXB, HOXC, and HOXD genes as well as from within the genes/gene regions of eight mouse models that exhibit NTDs. No significant findings were obtained for the tested markers.     

Neural tube defects and sex ratios

The sex ratio of 147 fetuses with presumed multifactorial neural tube defect (NTD) was studied. Overall, the ratio (males:females) was 0.73 with expected female excess. However, when the NTDs were subdivided according to the site of the lesion, the sex ratios varied. Total craniorachischisis, anencephaly with cervical spina bifida, holoacrania, and thoracic spina bifida showed a greater female excess than that overall; the sex ratio for meroacrania was close to unity, while that for low spinal lesions, particularly those involving the sacrum, showed an extreme bias towards males. These findings are related to the mode of formation of the neural tube. Females seem prone to defects of neurulation and males to defects in canalization. An explanation for these findings is suggested in possible sex differences in rate of early embryonic development.     

Prenatal ablation of nicotinic receptor alpha7 cell lineages produces lumbosacral spina bifida the severity of which is modified by choline and nicotine exposure

Lumbosacral spina bifida is a common debilitating birth defect whose multiple causes are poorly understood. Here, we provide the first genetic delineation of cholinergic nicotinic receptor alpha7 (Chrna7) expression and link the ablation of the Chrna7 cell lineage to this condition in the mouse. Using homologous recombination, an IRES-Cre bi-cistronic cassette was introduced into the 3' noncoding region of Chrna7 (Chrna7:Cre) for identifying cell lineages expressing this gene. This lineage first appears at embryonic day E9.0 in rhombomeres 3 and 5 of the neural tube and extends to cell subsets in most tissues by E14.5. Ablation of the Chrna7:Cre cell lineage in embryos from crosses with conditionally expressed attenuated diphtheria toxin results in precise developmental defects including omphalocele (89%) and open spina bifida (SB; 80%). We hypothesized that like humans, this defect would be modified by environmental compounds not only folic acid or choline but also nicotine. Prenatal chronic oral nicotine administration substantially worsened the defect to often include the rostral neural tube. In contrast, supplementation of the maternal diet with 2% choline decreased SB prevalence to 38% and dramatically reduced the defect severity. Folic acid supplementation only trended towards a reduced SB frequency. The omphalocele was unaffected by these interventions. These studies identify the Chrna7 cell lineage as participating in posterior neuropore closure and present a novel model of lower SB that can be substantially modified by the prenatal environment.     

Relationship between altered axial curvature and neural tube closure in normal and mutant (curly tail) mouse embryos

Neural tube defects, including spina bifida, develop in the curly tail mutant mouse as a result of delayed closure of the posterior neuropore at 10.5 days of gestation. Affected embryos are characterized by increased ventral curvature of the caudal region. To determine whether closure of the neuropore could be affected by this angle of curvature, we experimentally enhanced the curvature of non-mutant embryos. The amnion was opened in 9.5 day embryos; after 20 h of culture, a proportion of the embryos exhibited a tightly wrapped amnion with enhanced curvature of the caudal region compared with the control embryos in which the opened amnion remained inflated. Enhanced curvature correlated with a higher frequency of embryos with an open posterior neuropore, irrespective of developmental stage within the range, 27–32 somites. Thus, within this somite range, caudal curvature is a more accurate determinant for normal spinal neurulation than the exact somite stage. Enhanced ventral curvature of the curly tail embryo correlates with an abnormal growth difference between the neuroepithelium and ventral structures (the notochord and hindgut). We experimentally corrected this imbalance by culturing under conditions of mild hyperthermia and subsequently determined whether the angle of curvature would also be corrected. The mean angle of curvature and length of the posterior neuropore were both reduced in embryos cultured at 40.5°C by comparison with control embryos cultured at 38°C. We conclude that the sequence of morphogenetic events leading to spinal neural tube defects in curly tail embryos involves an imbalance of growth rates, which leads to enhanced ventral curvature that, in turn, leads to delayed closure of the posterior neuropore.