Human Spina Bifida Research Articles

Increased expression of Grainyhead-like-3 rescues spina bifida in a folate-resistant mouse model

Neural tube defects (NTDs), such as spina bifida, are common and severe birth defects in humans but the underlying causes are poorly understood. The pathogenesis and etiology of spina bifida in the curly tail mouse closely resemble defects in humans, providing a well-characterized model of NTDs. Grainyhead-like-3 (Grhl3), which encodes a transcription factor, was recently identified as a candidate gene for curly tail based on chromosomal location and the occurrence of spina bifida in Grhl3 null mice. However, the causative curly tail mutation has not been established, while the relationship between Grhl3 gene expression and the known cellular defect leading to NTDs in curly tail is unknown. Spina bifida in curly tail results from a cell type-specific proliferation defect in the hindgut endoderm, and we find that Grhl3 is expressed specifically in this tissue during the final stages of spinal neural tube closure in wild type embryos. Moreover, Grhl3 expression is diminished in the spinal region of neurulation-stage curly tail embryos. Curly tail mice do not carry a coding region mutation in Grhl3, however, we found a putative regulatory mutation upstream of the Grhl3 gene, which may be responsible for the expression deficit. In order to test the hypothesis that spina bifida in curly tail mice results from insufficient expression of Grhl3, we generated Grhl3-expressing curly tail mice by bacterial artificial chromosome-mediated transgenesis and demonstrated complete rescue of spina bifida. This study provides evidence for a critical role of diminished Grhl3 expression in causing spinal NTDs in the curly tail mouse model.

Screening for novel PAX3 polymorphisms and risks of spina bifida

PAX3 plays an important role in mammalian embryonic development. Known mutations in PAX3 are etiologically associated with Waardenburg syndrome and syndromic neural tube defects (NTDs). Mutations in the murine homologue, pax3, are responsible for the phenotype of splotch mice, in which nullizygotes are 100% penetrant for NTDs. The study sample included 74 infants with spina bifida (cases) and 87 nonmalformed infant controls. The conserved paired-box domain as well as the upstream genomic region of PAX3 were subjected to resequencing and those identified SNPs were evaluated as haplotypes. The associations of haplotypes for selected gene regions and the risks of spina bifida were further studied. Nineteen SNPs were observed; 15 observed in controls had been submitted to the National Center for Biotechnology Information (NCBI) database with allele frequencies. The PAX3 gene variant T-1186C (rs16863657) and its related haplotype, TCTCCGCCC of nine SNPs, were found to be associated with an increased risk of spina bifida, with an OR of 3.5 (95% CI: 1.2-10.0) among Hispanic Whites. Our analyses indicated that PAX3 SNPs were not strong risk factors for human spina bifida. However, additional follow-up of the PAX3 gene variant T-1186C (rs16863657) and its related haplotype, TCTCCGCCC, may be important in other populations.

Development of Lumbosacral Spina bifida: Three-Dimensional Computer Graphic Study of Human Embryos at Carnegie Stage Twelve

There is still some controversy as to whether sacral spina bifida in humans is the result of a defect of the primary or secondary neural tube. As somites are related to the development of vertebrae and the primary neural tube is related to the development of the spinal cord in embryos, it is very important to determine the number of somites in normal human embryos at the time of closure of the primary neural tube to understand the contribution of primary neural tube defects to the development of spina bifida. However, in the literature, the number of somites in stage 12 human embryos is still controversial. The aim of this study is to find the number of somites in human embryos at Carnegie stage 12. Four human embryos at Carnegie stage 12 were selected from the laboratory of the Congenital Anomaly Research Center in Japan. The neural tube and somites were reconstructed from their slices by a three-dimensional computer graphic reconstruction technique. The reconstructed embryos were examined from multidirectional magnified images. Thirty-three pairs of somites were present in all these reconstructed embryos. As the 33rd pair of somites corresponds to the fifth sacral segment, the presence of 33 pairs of somites at Carnegie stage 12 suggests that spina bifida develops from defects of the primary neural tube.

Control of neurulation by the nucleosome assembly protein-1–like 2

Neurulation is a complex process of histogenesis involving the precise temporal and spatial organization of gene expression. Genes influencing neurulation include proneural genes determining primary cell fate, neurogenic genes involved in lateral inhibition pathways and genes controlling the frequency of mitotic events. This is reflected in the aetiology and genetics of human and mouse neural tube defects, which are of both multifactorial and multigenic origin. The X-linked gene Nap1l2, specifically expressed in neurons, encodes a protein that is highly similar to the nucleosome assembly (NAP) and SET proteins. We inactivated Nap1l2 in mice by gene targeting, leading to embryonic lethality from mid-gestation onwards. Surviving mutant chimaeric embryos showed extensive surface ectoderm defects as well as the presence of open neural tubes and exposed brains similar to those observed in human spina bifida and anencephaly. These defects correlated with an overproduction of neuronal precursor cells. Protein expression studies showed that the Nap1l2 protein binds to condensing chromatin during S phase and in apoptotic cells, but remained cytoplasmic during G1 phase. Nap1l2 therefore likely represents a class of tissue-specific factors interacting with chromatin to regulate neuronal cell proliferation.

Animal models of neural tube defects

We reviewed the genetic variants and strains of mice that are used as models for neural tube defects (NTD) in humans. Over 40 genetic defects in mice cause obvious risk of NTD, but most are syndromic and many are lethal to embryos. Only a subset is similar to the common, nonsyndromic, genetically complex spina bifida or anencephaly in humans. The nonsyndromic variants that are potentially good models include homozygotes for spontaneous (Axd or Lp) or targeted (Apob, Macs, Mrp, or Trp53) mutations and five strains with spontaneous NTD of genetically complex cause, i.e., curly tail, SELH/Bc, NZW-xid, MT/HokIdr, and TO. Curly tail (1–5% exencephaly, 15–20% spina bifida) and SELH/Bc (15–20% exencephaly) are the best-understood developmental models for human spina bifida and anencephaly, but the genes are not yet known. The curly tail and Cart1 gene “knockout” models show that the defect leading to NTD may be in the supporting tissues, and not in the neural tube itself. The SELH/Bc model shows that there are compensatory mechanisms that can close the neural tube despite genetic deficiency of a normal closure mechanism. The Splotch mutations have been the most studied syndromic NTD in mice and are now known to be Pax3 gene mutations that model human Waardenberg syndrome, not common NTD. Heterogeneity of effective nutritional approaches to prevention is demonstrated by five genetically distinct models, each of which responds to a different nutrient. As in human anencephalics, an excess of females among exencephalics (of between 2–20-fold) is observed in seven mouse NTD genetically distinct models. Generally, strains with spontaneous NTD have a relatively high risk of NTD after exposure to the human teratogens, valproic acid, or retinoids. Nonsyndromic NTD in mice are genetically heterogeneous and often genetically complex, and we predict a similar genetic heterogeneity in human NTD. The genes contributing to the genetically complex NTD in mice, when identified, will provide candidate genes to test for association with human NTD risk. MRDD Research Reviews 1998;4:254–263. © 1998 Wiley-Liss, Inc.

Effect of supplemental folic acid on valproic acid-induced embryotoxicity and tissue zinc levels in vivo.

Valproic acid (VPA) is an anti-convulsant drug known to cause spina bifida in humans. Administration of the vitamin, folic acid, has been shown to decrease the recurrence and possibly also the occurrence of neural tube defects, primarily spina bifida, in humans. Additionally, treatment with a derivative (folinic acid) of folic acid has been reported to decrease the frequency of VPA-induced exencephaly in mice treated with the drug in vivo. A protective effect by folinic acid has not been observed in vitro. The purpose of this investigation was to reexamine the ability of folinic acid to decrease the incidence of VPA-induced neural tube defects in vivo. We also examined the effect of increased intake of folic acid on zinc levels in various maternal and embryonic tissues. Folinic acid, whether administered by intraperitoneal injection or in osmotic mini-pumps, did not decrease the number of mouse fetuses with VPA-induced exencephaly. Dietary supplementation with 10-20 times the daily required intake of folic acid in rodents also failed to decrease the embryotoxicity of VPA. Such dietary supplementation had no effect on zinc levels in maternal liver, brain, or kidney, nor in embryonic tissues. These results indicate that folic acid is not able to reverse the embryotoxicity induced by the anticonvulsant, that there is no apparent effect of high dietary folate intake on maternal or embryonic zinc levels and suggest that folate is probably not involved in the mechanism of VPA-induced embryotoxicity.

Interaction between undulated and Patch leads to an extreme form of spina bifida in double-mutant mice.

The aetiology of spina bifida involves genetic and environmental factors, which may be why major genes contributing to pathogenesis have not been identified. Here we report that undulated-Patch double-mutant mice have a phenotype reminiscent of an extreme form of spina bifida occulta in humans. This unexpected phenotype in double-mutant but not single-mutant mice shows that novel congenital anomalies such as spina bifida can result from interaction between products of independently segregating loci. This example of digenic inheritance may explain the often sporadic nature of spina bifida in humans.

Spinal Neural Tube Defects in the Curly Tail Mouse Mutant: Involvement of Retinoic Acid and its Nucleic Acid Receptors

Human neural tube defects (NTD) are among the commonest congenital malformations. The frequency of NTD is 1/1000 births in North America, Africa, Japan and Mongolia, and it rises to 7.6-8.6/1000 in south Wales and Northern Ireland. NTD also occur in 4.9% of all abortuses and so about 16/1000 of all conceptions are affected. The etiology of NTD is, however, still unknown. The mouse mutant curly tail (ct/ct) develops spinal NTD in 54% of cases, resulting from delayed closure of the posterior neuropore (PNP), and displays many similar features to those of NTD in humans. Thus, it provides a valid model for studying human spina bifida. Two previous observations suggest an involvement of retinoic acid (RA) and its mucleic receptors in closure of the PNP in mouse embryos. The effects of RA on development and growth of ct/ct embryos and on the expression pattern of nucleic retinoic acid receptor (RAR) were therefore studied. We found that maternal treatment with the low dose of 5 mg/kg RA decreases tlie incidence of spinal NTD by 50% in a stage-specific manner, without concomitant increase of tlie incidence of cranial NTD. The effect of RA is specific to tlie ct/ct phenotype: there is no other alteration in resorption rate, litter size, morphogenesis, growth or development. In situ hybridization using RAR-β and RAR-γ riboprobes and computerized image analysis of affected ct/ct embryos, which are destined to develop spinal NTD, reveals a down-regulation of RAR-β and RAR-γ expression in the hindgut endoderm and it tlie PNP region, respectively. The extent of reduction of RAR-γ transcripts abundance correlates well with the severity of delayed PNP closure. As early as 2 hours following RA treatment at 10 days 8 hours post coituni, RAR-β is upregulated specifically in the hindgut endoderm. The deficates expression of RAR-γ in the PNP region is also upregulated. By 6 hours after RA treatment, PNP length is significantly reduced. Three to four days later, the incidence of spinal NTD is decreased by 50%. This sequential event indicates that both the genesis and prevention of spinal NTD in the ct/ct mouse mutant involve retinoic acid and its mucleic acid receptors, RAR-β and RAR-γ.

Valproic acid‐induced spina bifida: A mouse model

Prenatal exposure to the antiepileptic drug valproic acid (VPA) has been associated with the formation of spina bifida aperta, meningocele, and meningomyelocele in the human. Until now, a direct relationship between VPA application and spina bifida has not been experimentally demonstrated. VPA was known only to induce exencephaly in mice, a defect of the anterior neural tube. Maximal sensitivity toward production of this defect was on day 8 of gestation (plug day = day 0). The closure of the posterior neuropore occurs later in the development of mice than the closure of the anterior neuropore. To investigate whether there is a direct relationship between VPA application during pregnancy and induction of spina bifida in mice, we administered various doses of the drug on day 9 of gestation, at three time intervals (at 0, 6, and 12 hr). This administration of VPA produced spina bifida aperta and spina bifida occulta in mice. High doses of VPA (3 x 450 and 3 x 500 mg/kg) induced a low rate of spina bifida aperta in the lumbosacral region. High incidences of spina bifida occulta, a less serious form of spina bifida, were induced with lower doses. This malformation was demonstrated in double-stained fetal skeletons by measurements of the distance between the cartilaginous ends of each vertebral arch. The occurrence of this defect and its localization was dose-dependent. The lumbar region was affected by all doses investigated (3 x 300, 3 x 350, 3 x 400, 3 x 450, and 3 x 500 mg/kg). The sacral/coccygeal region was affected additionally, but with higher doses (3 x 400, 3 x 450, and 3 x 500 mg/kg). A comparison of the results obtained with day 16 and 17 control fetuses showed that the pattern of gaps present in the lumbar and sacral region of the spinal cord in treated groups was drug-specific and not related to a developmental delay. Our results indicate that multiple administrations of VPA on day 9 of gestation in mice result in a low incidence of spina bifida aperta and a high incidence of spina bifida occulta, and provides a relevant model for the study of human spina bifida defects.

Teratogenic effects of valproate in the CD-1 mouse fetus.

Valproate sodium has been implicated in the production of spina bifida in humans; this article reports an animal model. Teratogenicity of valproate sodium was studied by oral administration of single doses of 225, 340, and 560 mg/kg to pregnant CD-1 mice on days 7 through 12 of gestation. All fetuses were examined on day 17. Treated fetuses demonstrated external malformations and a decrease in weight. The incidence of malformations was greater at the higher dosage levels of 340 mg/kg and 560 mg/kg, with a predominance of exencephaly, open eyelids, and gross skeletal defects. There was a significant increase in the resorption rate of the fetuses in the treated groups. There was also a significant increase in the malformations observed per litter and per live fetus population when compared with controls.

Are potatoes teratogenic for experimental animals?

AbstractThis study was designed to test in experimental animals the hypothesis (Renwick, 1972) that human spina bifida and anencephalus are causally related to maternal consumption of blighted potatoes. Gavaging of pregnant rats with blighted potatoes on days 5–11 or daily ip injections of solanine or glycoalkaloids (5–10 mg/kg) extracted from B5141‐6 potatoes, given on days 5–12 or 7–17 of gestation, resuited in minor skeletal and renal tract abnormalities, but failed to produce neural‐tube defects. Daily injections (days 5–12) of 20 and 40 mg/kg of the glycoalkaloid caused 100% fetal and maternal mortality, respectively. Daily ip injections of 5 mg/kg of solanine into New Zealand White rabbits on days 0–8 of gestation produced abortion in one‐third of the treated animals, 23% resorptions, but no teratogenesis. Single yolk‐sac injections of 5–20 mg/kg of solanine into 20–22 h incubated white Leghorn eggs produced 63–90% mortality within 72 h without significant teratogenesis. Diced B5141‐6 potatoes containing an average of 260 mg of solanine per kg of tuber fed ad libitum to time‐mated rhesus monkeys for a period of 25 days postcopulation produced no maternal toxicity; on the other hand single injections of 40 mg/kg of the glycoalkaloid extract and two injections of 20 mg/kg of solanine killed adults within 48 h of treatment. At autopsy nasal, oral, and periorbital hemorrhage was present; there was accumulation of serosanguinous pleural and peritoneal fluids and mild hepatic and splenic congestion. Examples of human toxicity and mortality from consuming potatoes with a high solanine content are reviewed.