Mouse mutants with neural tube closure defects and their role in understanding human neural tube defects

BACKGROUND: The number of mouse mutants and strains with neural tube closure defects (NTDs) now exceeds 190, including 155 involving known genes, 33 with unidentified genes, and eight "multifactorial" strains. METHODS: The emerging patterns of mouse NTDs are considered in relation to the unknown genetics of the common human NTDs, anencephaly, and spina bifida aperta. RESULTS: Of the 150 mouse mutants that survive past midgestation, 20% have risk of either exencephaly and spina bifida aperta or both, parallel to the majority of human NTDs, whereas 70% have only exencephaly, 5% have only spina bifida, and 5% have craniorachischisis. The primary defect in most mouse NTDs is failure of neural fold elevation. Most null mutations (>90%) produce syndromes of multiple affected structures with high penetrance in homozygotes, whereas the "multifactorial" strains and several null-mutant heterozygotes and mutants with partial gene function (hypomorphs) have low-penetrance nonsyndromic NTDs, like the majority of human NTDs. The normal functions of the mutated genes are diverse, with clusters in pathways of actin function, apoptosis, and chromatin methylation and structure. The female excess observed in human anencephaly is found in all mouse exencephaly mutants for which gender has been studied. Maternal agents, including folate, methionine, inositol, or alternative commercial diets, have specific preventative effects in eight mutants and strains. CONCLUSIONS: If the human homologs of the mouse NTD mutants contribute to risk of common human NTDs, it seems likely to be in multifactorial combinations of hypomorphs and low-penetrance heterozygotes, as exemplified by mouse digenic mutants and the oligogenic SELH/Bc strain.     

Ribonucleotide reductase subunit R1: a gene conferring sensitivity to valproic acid-induced neural tube defects in mice

Neural tube defects (NTDs), although prevalent and easily diagnosed, are etiologically heterogeneous, rendering mechanistic interpretation problematic. To date, there is evidence that mammalian neural tube closure (NTC) initiates and fuses intermittently at four discrete locations. Disruption of this process at any of these four sites may lead to a region-specific NTDs, possibly arising through closure site-specific genetic mechanisms. Although recent efforts have focused on elucidating the genetic components of NTDs, a void persists regarding gene identification in closure site-specific neural tissue. To this end, experiments were conducted to identify neural tube closure site-specific genes that might confer regional sensitivity to teratogen-induced NTDs. Using an inbred mouse strain (SWV/Fnn) with a high susceptibility to VPA- induced NTDs that specifically targets and disrupts NTC between the prosencephalon and mesencephalon region (future fore/midbrain; neural tube closure site II), we identified a VPA-sensitive closure site II-specific clone. Sequencing of this clone from an SWV neural tube cDNA library confirmed that it encodes the r1 subunit of the cell cycle enzyme ribonucleotide reductase (RNR). The abundance of rnr-r1 mRNA was significantly increased in response to VPA drug treatment. This upregulated expression was accompanied by a significant decrease in cellular proliferation in the closure site II neural tube region of the embryos, as determined by ELISA cellular proliferation assays performed on BrdU-pulsed neuroepithelial cells in vivo. We hypothesize that rnr-r1 plays a critical role in the development of VPA-induced exencephaly.     

Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)-based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.     

Maternal diet alters exencephaly frequency in SELH/Bc strain mouse embryos

BACKGROUND: The SELH/Bc mouse inbred strain, with a high frequency of nonsyndromic, genetically-multifactorial exencephaly, is a model for human cranial neural tube defects (NTDs). Maternal diet affects risk of human NTDs. METHODS: Exencephaly frequencies in SELH/Bc embryos were compared in 8 studies in which dams were fed alternative commercial Purina diets (5015 and 5001) or semisynthetic diets, and in several studies in which maternal diet was supplemented with a specific nutrient, either in drinking water or food before and during pregnancy, or by intraperitoneal injection on E7 and/or E8. RESULTS: The exencephaly frequency in SELH/Bc embryos was 2- to 8-fold higher when the dams were fed Purina 5015 (averaging 23% exencephaly) or a semisynthetic diet modeled on Purina 5015 (averaging 28%) or NIH-31 standard diet (23%), compared with Purina 5001 (averaging 7%). The exencephaly frequency remained high (41%) on a semisynthetic diet modeled on Purina 5001. The exencephaly frequency was not reduced significantly by maternal supplementation with folic acid, nor with each of zinc, methionine, niacin, brewers' yeast, riboflavin, vitamin B12, or inositol. Nor was it reduced by maternal diets with supplemental methyl donors and cofactors or with reduced fat. CONCLUSIONS: The frequency of exencephaly in SELH/Bc embryos is strongly influenced by a specific unidentified aspect of the commercial ration Purina 5001 that prevents 55-85% of exencephaly in SELH/Bc embryos, when directly compared with an alternative commercial ration Purina 5015 or its semisynthetic mimic. This strong maternal diet effect on NTD frequency may point to novel nutritional approaches to prevention of human NTDs.     

Normal mouse strains differ in the site of initiation of closure of the cranial neural tube

The scanning electron microscopic study of day 9 embryos reported here documents differences among normal mouse strains in morphology of cranial neural tube closure. The site of initiation of contact and fusion of the cranial neural folds, previously defined as Closure 2 (Macdonald et al., '89), is located in the region of the junction between the forebrain (prosencephalon) and midbrain (mesencephalon) in three normal strains: LM/Bc, AEJ/RkBc, and ICR/Bc. However in a fourth normal strain, SWV/Bc, Closure 2 is initiated much further rostral, in the prosencephalic region. In addition, the anterior neuropore, rostral to Closure 2, closes late in ICR/Bc embryos, relative to the posterior progress of development of the Closure 2 seam. Initiation of closure from the most rostral end of the neural tube (Closure 3) appears to be relatively delayed in ICR/Bc embryos. We hypothesize that the observed genetic polymorphism in location of the first site of fusion between the cranial neural folds in normal mouse embryos may be one basis for differences among normal strains in liability to exencephaly induced by teratogens.     

Mini-review: toward understanding mechanisms of genetic neural tube defects in mice.

We review the data from studies of mouse mutants that lend insight to the mechanisms that lead to neural tube defects (NTDs). Most of the 50 single-gene mutations that cause neural tube defects (NTDs) in mice also cause severe embryonic-lethal syndromes, in which exencephaly is a nonspecific feature. In a few mutants (e.g., Trp53, Macs, Mlp or Sp), other defects may be present, but affected fetuses can survive to birth. Multifactorial genetic causes, as are present in the curly tail stock (15-20% spina bifida), or the SELH/Bc strain (15-20% exencephaly), lead to nonsyndromic NTDs. The mutations indicate that "spina bifida occulta," a dorsal gap in the vertebral arches over an intact neural tube, is usually genetically and developmentally unrelated to exencephaly or "spina bifida" (aperta). Almost all exencephaly or spina bifida aperta of genetic origin is caused by failure of neural fold elevation. The developmental mechanisms in genetic NTDs are considered in terms of distinct rostro-caudal zones along the neural folds that likely differ in mechanism of elevation. Failure of elevation leads to: split face (zone A), exencephaly (zone B), rachischisis (all of zone D), or spina bifida (caudal zone D). The developmental mechanisms leading to these genetic NTDs are heterogeneous, even within one zone. At the tissue level, the mutants show that the mechanism of failure of elevation can involve, e.g., (1) slow growth of adjacent tethered tissue (curly tail), (2) defective forebrain mesenchyme (Cart1 or twist), (3) defective basal lamina in surface ectoderm (Lama5), (4) excessive breadth of floorplate and notochord (Lp), (5) abnormal neuroepithelium (Apob, Sp, Tcfap2a), (6) morphological deformation of neural folds (jmj), (7) abnormal neuroepithelial and neural crest cell gap-junction communication (Gja1), or (8) incomplete compensation for a defective step in the elevation sequence (SELH/Bc). At the biochemical level, mutants suggest involvement of: (1) faulty regulation of apoptosis (Trp53 or p300), (2) premature differentiation (Hes1), (3) disruption of actin function (Macs or Mlp), (4) abnormal telomerase complex (Terc), or (5) faulty pyrimidine synthesis (Sp). The NTD preventative effect of maternal dietary supplementation is also heterogeneous, as demonstrated by: (1) methionine (Axd), (2) folic acid or thymidine (Sp), or (3) inositol (curly tail). The heterogeneity of mechanism of mouse NTDs suggests that human NTDs, including the common nonsyndromic anencephaly or spina bifida, may also reflect a variety of genetically caused defects in developmental mechanisms normally responsible for elevation of the neural folds.     

Neural tube development requires the cooperation of p53- and Gadd45a-associated pathways

BACKGROUND: Numerous genetically engineered mouse models for neural tube defects (NTDs) exist, and some of the implicated proteins are functionally related. For example, the growth arrest and DNA damage-inducible protein Gadd45a and tumor suppressor p53 are functionally similar, and both are involved in neural tube development (Gadd45a- and Trp53-null embryos show low levels of exencephaly). To assess their roles in neural tube development, we generated double-null mice from Gadd45a- and Trp53-null mice, as well as from cyclin-dependent kinase inhibitor (Cdkn1a) (p21)-null and xeroderma pigmentosum group C (XPC)-null mice that do not show spontaneous exencephaly. METHODS: Gadd45a-, Trp53-, Cdkn1a-, and XPC-null mice were crossed to generate several double-null mouse models. Embryos (embryonic day [ED] 16-18) from the single- and double-null crosses were scored for NTDs. RESULTS: Deletion of both Gadd45a and Trp53 in mice increased exencephaly frequencies compared to the deletion of either single gene (34.0% in Gadd45a/Trp53-null compared to 8.4% and 9.1% in the Gadd45a- and Trp53-null embryos, respectively). Furthermore, although deletion of another p53-regulated gene, Cdkn1a, is not associated with exencephaly, in conjunction with Gadd45a deletion, the exencephaly frequencies are increased (30.5% in the Gadd45a/Cdkn1a-null embryos) and are similar to those in the Gadd45a/Trp53-null embryos. Although XPC deletion increased exencephaly frequencies in Trp53-null embryos, XPC deletion did not increase the exencephaly frequencies in Gadd45a-null embryos. CONCLUSIONS: The increased genetic liability to exencephaly in the Gadd45a/Trp53- and Gadd45a/Cdkn1a-null embryos may be related to the disruption of multiple cellular pathways associated with Gadd45a and p53.     

A systems‐based approach to investigate dose‐ and time‐dependent methylmercury‐induced gene expression response in C57BL/6 mouse embryos undergoing neurulation

BACKGROUND: Aberrations during neurulation due to genetic and/or environmental factors underlie a variety of adverse developmental outcomes, including neural tube defects (NTDs). Methylmercury (MeHg) is a developmental neurotoxicant and teratogen that perturbs a wide range of biological processes/pathways in animal models, including those involved in early gestation (e.g., cell cycle, cell differentiation). Yet, the relationship between these MeHg‐linked effects and changes in gestational development remains unresolved. Specifically, current information lacks mechanistic comparisons across dose or time for MeHg exposure during neurulation. These detailed investigations are crucial for identifying sensitive indicators of toxicity and for risk assessment applications. METHODS: Using a systems‐based toxicogenomic approach, we examined dose‐ and time‐dependent effects of MeHg on gene expression in C57BL/6 mouse embryos during cranial neural tube closure, assessing for significantly altered genes and associated Gene Ontology (GO) biological processes. Using the GO‐based application GO‐Quant, we quantitatively assessed dose‐ and time‐dependent effects on gene expression within enriched GO biological processes impacted by MeHg. RESULTS: We observed MeHg to significantly alter expression of 883 genes, including several genes (e.g., Vangl2, Celsr1, Ptk7, Twist, Tcf7) previously characterized to be crucial for neural tube development. Significantly altered genes were associated with development cell adhesion, cell cycle, and cell differentiation–related GO biological processes. CONCLUSIONS: Our results suggest that MeHg‐induced impacts within these biological processes during gestational development may underlie MeHg‐induced teratogenic and neurodevelopmental toxicity outcomes. Birth Defects Res (Part B) 89:188–200, 2010. © 2010 Wiley‐Liss, Inc.     

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     

Studies of the effect of retinoic acid on anterior neural tube closure in mice genetically liable to exencephaly

Previously we have shown that all SELH/Bc mouse embryos close their anterior neural tubes by an abnormal mechanism and that 10-20% of SELH/Bc embryos are exencephalic. The purposes of these studies were (1) to observe the effects of retinoic acid on the frequency of exencephaly in SELH/Bc embryos; (2) to compare the SELH/Bc response with those of normal strains and of other neural tube mutants; and (3) to compare, between SELH/Bc and a normal strain (SWV/Bc), the effects of retinoic acid on morphology of the closing anterior neural tube. SELH/Bc was more liable to retinoic acid-induced exencephaly than were normal strains. After maternal treatment with 5 mg/kg retinoic acid on day 8.5 of gestation, 53% of SELH/Bc embryos had exencephaly, compared with 22% in ICR/Bc and 14% in SWV/Bc. When these results were transformed according to the assumptions of the developmental threshold model, the effects of genotype and retinoic acid appeared to be additive. Similar treatment on day 9 or 10 of gestation had little or no effect on the frequency of exencephaly in SELH/Bc mice. These results are similar to the reported responses of the curly-tail and Splotch mutants, where frequencies of spina bifida but not exencephaly were decreased. This pattern suggests that studies of effects of periconceptional vitamin treatment on risk of human neural tube defects should consider anencephaly and spina bifida separately. The study comparing the morphology of anterior neural tube closure in SELH/Bc and normal SWV/Bc embryos showed that retinoic acid delays the elevation of the mesencephalic neural folds. This results in a "stalling" of many embryos in the first steps of neural tube closure, with their neural folds remaining convex and splayed wide apart. The delay in fold elevation was superimposed on the different closure patterns of the two strains. The overall conclusion is that there is no nonadditive interaction in the parameters studied between retinoic acid treatment and the SELH/Bc genotype.     

Developmental study of neural tube closure in a mouse stock with a high incidence of exencephaly

About 17% of embryos and fetuses in the SELH/Bc mouse stock have the anterior neural tube defect, exencephaly. No other malformations are seen. The genetic liability to exencephaly was shown to be probably genetically fixed in the SELH/Bc stock. This means that SELH/Bc embryos with successful neural tube closure are genetically the same as exencephalics. Females were significantly more likely to be affected than males (66% females). The pattern of morphological developmental events during anterior neural tube closure on days 8 and 9 of gestation was compared among 322 ICR/Bc (normal), 304 SWV/Bc (normal), and 265 SELH/Bc embryos. Anterior neural tube closure was found to follow a strikingly different pattern in almost all SELH/Bc embryos than in either of the normal strains or in previous published studies. SELH/Bc embryos lack the initial contact between the anterior folds in the posterior prosencephalon/anterior mesencephalon region (Closure 2). In spite of this, all but 17% manage to close the anterior neural tube by extending caudally the later occurring normal anterior zone of contact and fusion at the most rostral aspect of the prosencephalon (Closure 3) through the region of Closure 2 to meet the zone of closure of the rhombencephalon, Closure 4. Anterior neural tube closure was completed late, and in some SELH/Bc embryos, elevation and fusion in the mesencephalon did not occur at all. In histological sections of six- and eight-somite embryos, elevated numbers of pyknotic cells in the neuroepithelium and mesenchyme, and elevated numbers of unstained inclusions in the neuroepithelium were found; but their relationship, if any, to the abnormal pattern of neural tube closure is not clear.     

Quantitative analysis of s-adenosylmethionine and s-adenosylhomocysteine in neurulation-stage mouse embryos by liquid chromatography tandem mass spectrometry

The potential importance of the methylation cycle during embryonic development necessitates the establishment of methodology to detect alterations in the relative abundance of s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) in an embryonic experimental system. We have developed a precise and sensitive method for measurement of SAM and SAH based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in single neurulation-stage mouse embryos. Use of a penta-fluorinated high-performance liquid chromatography (HPLC) stationary phase gave enhanced sensitivity due to optimal ionisation in organic mobile phase and increased retention time compared to standard reversed-phase separation. Calibration curves suitable for the analysis of neurulation-stage mouse embryos (SAM 0.02-25.0microM, SAH 0.01-10.0microM) were linear (r(2)>0.997) with limits of detection for SAM and SAH of 10 and 2.5nmol/L, respectively.     

Phactr4 regulates neural tube and optic fissure closure by controlling PP1-, Rb-, and E2F1-regulated cell-cycle progression

Here we identify the humpty dumpty (humdy) mouse mutant with failure to close the neural tube and optic fissure, causing exencephaly and retinal coloboma, common birth defects. The humdy mutation disrupts Phactr4, an uncharacterized protein phosphatase 1 (PP1) and actin regulator family member, and the missense mutation specifically disrupts binding to PP1. Phactr4 is initially expressed in the ventral cranial neural tube, a region of regulated proliferation, and after neural closure throughout the dorsoventral axis. humdy embryos display elevated proliferation and abnormally phosphorylated, inactive PP1, resulting in Rb hyperphosphorylation, derepression of E2F targets, and abnormal cell-cycle progression. Exencephaly, coloboma, and abnormal proliferation in humdy embryos are rescued by loss of E2f1, demonstrating the cell cycle is the key target controlled by Phactr4. Thus, Phactr4 is critical for the spatially and temporally regulated transition in proliferation through differential regulation of PP1 and the cell cycle during neurulation and eye development.     

Phenylalanine and its metabolites induce embryopathies in mouse embryos in culture

The aim of this study was to determine the teratogenicity of phenylalanine (Phe) and Phe metabolites in neurulating mouse embryos. Therefore, the system of whole embryo culture was employed and D9 (neurulating) mouse embryos were exposed to Phe, phenylethylamine (PEA), phenylpyruvic acid (PPA), phenylacetic acid (PAA), 2-OH phenylacetic acid (2-OH PAA), and phenyl-lactic acid (PLA) at concentrations ranging from 0.01 mM to 10 mM for 24 hours. After 24 hours, embryos were examined for morphological abnormalities and protein content by the Lowry method. Phe at 1 and 6 mM concentrations was not teratogenic; however, 10 mM inhibited cranial neural tube closure in 82% of the embryos. PEA was the most toxic factor and concentrations of 1 and 10 mM were embryo-lethal, whereas neural tube closure defects (NTDs) were observed in 67% of the embryos at 0.1 mM. 2-OH PAA was the second most toxic metabolite with concentrations of 1 and 10 mM producing NTDs in 10 and 100% of the embryos, respectively. PLA and PAA produced no NTDs at concentrations of 1 mM, 60% at 5 mM, and 100% at 10 mM. Finally, PPA produced approximately 50% NTDs at both 1 mM and 10 mM concentrations. PLA, PAA, 2-OH PAA, and PPA produced a significant reduction in embryonic protein, and PEA and 2-OH PAA reduced yolk sac protein values. PEA, 2-OH PAA, PPA, PAA, and PLA also produced craniofacial abnormalities, i.e., incomplete expansion of the forebrain, collapse of the optic vesicle, and hypoplasia of the mandible and/or the maxilla.(ABSTRACT TRUNCATED AT 250 WORDS)     

The emerging role of epigenetic mechanisms in the etiology of neural tube defects

The molecular requirements for neural tube closure are complex. This is illustrated by the occurrence of neural tube defects (NTDs) in many genetic mouse mutants, which implicate a variety of genes, pathways and cellular functions. NTDs are also prevalent birth defects in humans, affecting around 1 per 1,000 pregnancies worldwide. In humans the causation is thought to involve the interplay of fetal genes and the effect of environmental factors. Recent studies on the etiology of human NTDs, as well as analysis of mouse models, have raised the question of the possible involvement of epigenetic factors in determining susceptibility. A consideration of potential causative factors in human NTDs must now include both alterations in the regulation of gene expression, through mutation of promoter or regulatory elements and the additional analysis of epigenetic regulation. Alterations in the epigenetic status can be directly modified by various environmental insults or maternal dietary factors.Key words: neural tube defects, diet, folic acid, epigenome, epigenetic regulation, methylation, chromatin, histones, acetylation     

Using genomewide mutagenesis screens to identify the genes required for neural tube closure in the mouse

BACKGROUND: Neural tube closure is a critical embryological process that requires the coordination of many molecular and cellular events. Only recently has the molecular basis of the cell movements that drive neural tube closure begun to be elucidated. This has been accomplished in part due to the analysis of a growing number of genetically targeted and naturally occurring mouse mutant strains that have neural tube defects (NTDs). Currently there are more than 100 genes that when mutated result in NTDs in the mouse. Yet only approximately 10% of genes in the mouse genome have been mutated and their gross phenotype analyzed, suggesting that only a small percentage of the genes that can cause NTDs have been identified. METHODS: In order to more systematically and fully understand the genetic basis of neural tube closure and to begin to define the molecular pathways that direct this key embryonic event, our laboratories have undertaken a forward genetic screen in mice. From this we hope to gain a better understanding of the regulation of this complex morphogenic processes. CONCLUSIONS: The mouse provides a good model for human neural tube closure, and therefore the information gained from generating novel mouse models of NTDs will help to predict the genes responsible for human NTDs and provide experimental evidence for how they function.