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.     

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.     

Cranial effects of retinoic acid in the loop-tail (Lp) mutant mouse

The effects of retinoic acid (RA) on the manifestation and nature of neural tube defects (NTD) in heterozygous embryos of mutant mice carrying the gene loop-tail (Lp) and in normal (+/+) littermates and embryos from normal homozygous matings were compared with NTD that occur in untreated abnormal homozygous (Lp/Lp) embryos. A single intraperitoneal dose (5 mg/kg) of RA administered at 9 AM or 3 PM on day 8 of gestation induced NTD in +/+ as well as Lp/+ embryos removed on day 12 of gestation. All of the NTD were confined to the brain and consisted of exencephaly involving the diencephalon, mesencephalon, and metencephalon. In neither phenotype (Lp/+; +/+) was the massive exencephaly and myeloschisis characteristic of untreated Lp/Lp embryos produced; thus, it is possible that the teratogenic mechanisms of RA-induced defects and of Lp-induced defects may differ.     

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.     

The curly-tail mouse: an experimental model for human neural tube defects

Many aspects of the open lesions of the neural tube observed in the curly-tail mice are similar to those seen in man. In the mouse, the cause of the defects is a recessive gene, the expression of which is modified by the rest of the genome. A marked interaction between genetic and environmental factors was also observed in this strain of mice, thus suggesting that it represents a useful model to investigate the origin of the neural tube defects (NTD) in man. In the mutant strain, the interaction between the curly-tail gene and the environmental factors has been investigatigated using known teratogenic compounds, as well as hormones and vitamins, which it was hoped would prevent the malformations. An intriguing result was to observe that, while, as expected, excess of vitamin A increases the incidence of NTD, low doses, given at a particular stage of gestation, prevent the manifestation of the abnormalities.To investigate further the interaction between genetic and environmental factors and to provide some insight into the mechanisms underlying NTD in man, the effects of Trypan blue, hydroxyurea, progesterone, cortisone and vitamin E were also investigated in curly-tail mice and the results reported in this review.     

Comparison of the incidence of 5-azacytidine-induced exencephaly between MT/HokIdr and Slc:ICR mice.

The incidence of 5-azacytidine-induced exencephaly was compared between MT/HokIdr strain (MT) and Slc:ICR strain (ICR) mice. MT mice have a genetic predisposition for exencephaly, but ICR mice do not. Pregnant mice were given 5-azacytidine (1 mg/kg to 100 micrograms/kg) injected intraperitoneally on Day 7.5 of gestation (vaginal plug day = Day 0.5), and fetuses were observed for external malformations on Day 18.5 of gestation. One hundred micrograms/kg 5-azacytidine induced exencephaly in MT mice but not in ICR mice, and 1 mg/kg 5-azacytidine resulted in resorptions in MT mice but caused exencephaly in ICR mice. These results indicated that MT mice had 10-fold more sensitivity to 5-azacytidine than ICR mice. It seems likely that less than effective doses of teratogens for animals without genetic predispositions are still effective in inducing malformations in animals with a genetic predisposition for malformations. When 4-somite-stage embryos of both MT and ICR mice were cultured in rat serum supplemented with 5-azacytidine, 0.02 micrograms/ml 5-azacytidine induced the failure of closure of cephalic neural tube in MT embryos but not in ICR embryos, and 0.2 micrograms/ml 5-azacytidine induced severe growth retardation in MT embryos but in ICR embryos it only induced embryos with smaller heads and fewer somites than in control. These results indicated that MT mouse embryos in culture also had a 10-fold-increased sensitivity to 5-azacytidine compared with ICR mouse embryos, suggesting maternal effects play no significant role in their increased sensitivity to 5-azacytidine.     

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.     

Cyclophosphamide teratogenesis: evidence for compensatory responses to induced cellular toxicity

Cyclophosphamide (CP) administered ip to pregnant mice on day 10 of gestation (day of plug = day 0) is teratogenic (exencephaly, cleft palate, and limb malformations) at 20 mg/kg and embryolethal at higher doses. In the present study, CP was administered at 1, 5, 10, or 20 mg/kg on day 10 of gestation. Embryos were removed at 8 and 28 hr postdosing, and two embryos from each litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death. The remaining embryos were frozen and forelimb buds subsequently removed for flow cytometric (FCM) analysis of the cellular DNA synthetic cycle. Additional litters were examined near term (day 17) for morphological abnormalities; these data were correlated with embryonic toxicity as detected by NBS staining and FCM analysis. Only the highest dose produced malformations. In marked contrast, a dose-related increase in the percentage of limb bud cells in the S (DNA synthetic) phase of the cell cycle was detectable at all doses. Inhibition of DNA synthesis was detected at all doses 8 hr post exposure and persisted through 28 hr for doses greater than or equal to 10 mg/kg. NBS staining indicated increased cell death in the alar plate of the neural tube 28 hr after exposure to 10 mg/kg CP and generally increased cell death in areas of rapid cell proliferation throughout the embryo at 20 mg/kg. The absence of an overt teratogenic response at dose levels that produced significant perturbation of the cell cycle indicates that a measure of embryonic damage can be compensated for or repaired. The implications of these findings for the existence of thresholds in developmental toxicity are discussed.     

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.     

Maternal methionine supplementation promotes the remediation of axial defects in Axd mouse neural tube mutants.

The Axd (axial defects) mouse model system (Essien et al., Teratology 42:183-194, '90) is characterized by a dominant mutation which causes posterior open neural tube defects (NTD) and a variety of tail anomalies (curly tails, or CT). Repeated backcrosses to BALB/cByJ mice have resulted in a 50% increase in Axd penetrance among neonates of heterozygous matings and loss of a correlation with maternal tail phenotype. Analysis of D12-D18 embryos from Axd/+ x Axd/+ matings indicates that soft tissues can superficially heal over some lesions from open NTD and that some curly tails can straighten (macroscopically) as gestation proceeds. Similarly, in embryos of Axd/+ x BALB crosses, there is remediation of approximately 33% of the tail flexion defects by birth. Numerous studies show that maternal nutritional status can affect the development of the neural tube and related axial structures. One nutrient of special interest is methionine, which is required for neurulation in cultured rat embryos (Coelho et al., J. Nutr. 119:1716, '89). Thus, the major question addressed by this study was whether supplemental methionine administered to Axd/+ dams crossed to Axd/+ males would alter the prenatal expression of the gene. When given IP (70 mg/kg) on D8 and D9, methionine resulted in a 41% reduction (from 29% to 17%) in the incidence of NTD in D 14 embryos (P less than 0.01).     

Effect of antiepileptic drugs valproic acid, carbamazepine and ethosuccimide on exploratory behaviour in mice

Valproic acid in 100, 200 and 400 mg/kg doses produced a significant dose dependent decrease in exploratory behaviour, tested as number of head dips on the hole board. In the open field test, control mice entered less number of peripheral squares and more number of central squares on day 4 as compared to day 1 of the test. In the lower doses (100 and 200 mg/kg) valproic acid increased central square entries on day 1 with significant decrease by all doses on subsequent days indicating inhibition of exploratory behaviour. However, in peripheral square entry they followed the same pattern as control mice. Neither carbamazepine (10 and 20 mg/kg) nor ethosuccimide (100 and 200 mg/kg) affected exploratory behaviour in either the hole board or open field test.     

Vinyl Chloride Monomer (VCM) Induces High Occurrence of Neural Tube Defects in Embryonic Mouse Brain During Neurulation

The aim of this study was to explore the direct embryonic teratogenicity of vinyl chloride monomer (VCM), especially the toxic effects on the early development of the nervous system and its underlying mechanisms. Pregnant mice at embryonic day 6.5 (E6.5) were injected with different doses of VCM (200, 400 and 600 mg/kg) and embryos were harvested at E10.5. Our results showed that doses higher than 400 mg/kg of VCM increased the incidence of malformed embryos, especially the neural tube defects (NTDs). In addition, high-dose of VCM decreased mitotic figure counts in the neuroepithelium and enhanced the percentage of cells in G0/G1 phase, while they were reduced in S phase. The more VCM was injected into mice, the fewer positive PCNA cells were seen and the more positive TUNEL cells were observed in the neuroepithelium. Moreover, significant increases in the levels of caspase-3 protein were observed in NTD embryos. Our results demonstrate that during early pregnancy, exposure to doses higher than 400 mg/kg of VCM increases the incidence of malformations and particularly the rate of NTDs. High-dose of VCM inhibits the proliferation of neural cells and induces cell apoptosis, leading to an imbalance in the ratio of proliferation and apoptosis. Meanwhile, the apoptosis of neuroepithelial cells might be accelerated by the activation of the caspase-3 pathway, and it might be a reason for NTDs.     

Strain differences in the teratogenicity induced by sodium valproate in cultured mouse embryos

Strain differences in the teratogenicity of valproic acid (VPA) have been reported in mice. Finnell and Chernoff (Proc. Grnwd. Genet. Ctr. 5:162-163, 1985) showed that 300 mg/kg of VPA twice a day on days 6-8 of gestation induced exencephaly in 82% of SWV embryos but in 0% of C57BL/6J embryos. In the present experiment, we have collected similar results and investigated this strain difference using whole embryo culture in an attempt to determine whether maternal or embryonic factors are responsible for the difference. Mouse embryos were explanted on day 8.5 (plug day 0), and embryos at the 6-8-somite stage were cultured for 48 hours in rat serum containing various doses of sodium valproate (NaVP). All the embryos died within 24 hours with 4.5-mM and higher doses of NaVP in C57BL/6NCr1BR (C57) and with 3.0-mM and higher doses in SWV. Unfused brain folds were recognized in embryos treated with 3.0-mM and higher doses in C57, and with 1.0-mM and higher doses in SWV. Irregular somite formation was observed in many embryos treated with 1.6-mM and higher doses in C57 and with 1.0-mM and higher doses in SWV. These results indicate that SWV embryos have 1.5-3 times the sensitivity of C57 embryos to the embryolethal and teratogenic effects of NaVP. Furthermore, the results suggest that the basis of the strain difference resides within the embryo rather than the mother.     

Retinoic acid-induced selective mortality of splotch-delayed mouse neural tube defect mutants

The allelic loci splotch (Sp) and splotch-delayed (Spd) cause neural tube defects (NTDs) in mice homozygous for either of these genes. The polymorphic enzyme isocitrate dehydrogenase (Idh-1) in conjunction with a recombination suppressor was used as a genetic marker to identify embryos homozygous for these alleles. A split dose of all-trans retinoic acid (RA) totalling 5.0 mg/kg administered on gestation day 9/15 and 9/18 (days/h) significantly reduced the frequencies of NTD and of mutant genotypes in marked Spd embryos examined on day 16 without significantly increasing the resorption frequency. There was a nonsignificant decrease in the frequencies of NTD and mutant genotypes in embryos examined on day 11 of gestation. Thus, retinoic acid treatment was associated with selective mortality of the homozygous Spd mutants. No evidence of selective mortality was observed in RA-treated Sp embryos.     

The effects of 5-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo

The effects of 500 and 300 mg/kg bromodeoxyuridine (BUdR) on the process of fusion of the neural folds were tested after injection into pregnant mice on day 8 of gestation (192 hours postcoitum). Various doses of the natural nucleoside, thymidine (TdR), were also tested. Both doses of BUdR retarded growth to the same extent, but only the larger dose caused neural tube defects in 28.8% of embryos. Treatment with the larger dose also caused extensive cell necrosis to appear in the neuroepithelium of the neural folds between 12 and 15 hours after treatment. No changes were detectable with the light microscope up to this time. Measurement of the cell generation time in treated and control embryos indicated that the BUdR prolonged the cycle by about 2 hours and that the dying cells were in the second DNA synthetic phase following incorporation of the analog. Treatment with the smaller dose of BUdR caused minimal cell necrosis. This was taken as evidence for the importance of cell necrosis in the pathogenesis of BUdR-induced neural tube defects. Treatment with excess TdR did not cause either neural tube defects or cell necrosis, and a dose of TdR equimolar with the large dose of BUdR (400 mg/kg TdR) did not retard growth. Doses of 800 and 1,200 mg/kg TdR retarded growth to the same extent as BUdR. The administration of an equimolar amount of TdR, along with the teratogenic dose of BUdR, prevented the occurrence of cell necrosis and neural tube defects. When treatments were given on day 9 of gestation, 500 mg/kg BUdR caused cell necrosis in the neuroepithelium about 15 hours after treatment but no neural tube defects were produced by day 9 after treatment. It is suggested that in this case cell necrosis occurred too late to interfere with neural fold fusion. It was concluded that the ability of BUdR to cause exencephaly in mouse embryos was due to cell necrosis in the neuroepithelium.     

Interference of Mycotoxins with Prenatal Development of the Mouse

The prenatal effects of mycotoxins were investigated in GBA mice given by stomach tube a single dose of either aflatoxin B1 (4 mg/kg), ochratoxin A (8 mg/kg) or zearalenone (20 mg/kg) on pregnancy day 8 or 9. Aflatoxin caused foetal anomalies (exencephaly, open eyes, and protrusion of intestines) after exposure on gestation day 8 but not on day 9. The effects (increased prenatal mortality, reduced foetal growth, and a wide variety of malformations) caused by ochratoxin were much more severe and occurred after treatment on either of the 2 days of gestation. Among the spectrum of malformations, predominantly involving the craniofacial complex and the axial skeleton, the most striking was the total aplasia/dysplasia of the upper facial structures. These defects were always accompanied by exencephaly and anophthalmia. Zearalenone caused no effects. It is concluded that of the 3 mycotoxins screened with the technique used, ochratoxin is the most potent teratogen in mice.     

Exencephaly and axial skeletal dysmorphogenesis induced by maternal exposure to cadmium in the mouse

To investigate the axial skeletal dysmorphogenesis associated with exencephaly and facial abnormalities, two doses of cadmium chloride (4 mg/kg and 6 mg/kg) were administered subcutaneously to MF1 mice on day 7 of gestation (sperm-positive day = day 0). Fetuses were collected on day 18. Gross examination revealed a very high incidence of cranioschisis aperta with exencephaly, maxillary and mandibular hypoplasia, low-set microtia, edema, and growth retardation of fetuses in both treatment groups. Alizarin red S-stained and cleared skeletal preparations of these embryos revealed hypoplasia of the premaxilla, maxilla, nasal bone, zygoma, and mandible of the facial skeleton. The orbital plate represented the frontal bone. The vault of the skull was conspicuously absent. In cranioschisis, the exoccipitals had splayed and fused with the atlas. The basicranial bones were hypoplastic and crowded, thus reducing the cranial cavity. The vertebral bodies were more affected than the arches. Hemivertebrae and longitudinal fusion of centra and arches were also noted. The ribs were usually rudimentary. Agenesis, fusion, and forking of ribs were frequently observed. The sternebrae were rudimentary, bipartite, or longitudinally fused. These data clearly establish the association between neural tube and axial mesodermal abnormalities and emphasize the interdependence of the neurectoderm and mesoderm in normal morphogenesis.     

Teratogenic effects of nigericin, a carboxylic ionophore

Nigericin (Na+ salt) was given intraperitoneally at doses of 5.0 or 7.0 mg/kg on one of gestation days 7-12 to pregnant CD-1 mice. Additional mice were injected ip with 2.5 mg/kg on day 11 or 12 only. Injections on single gestation days reduced fetal growth and increased prenatal deaths. Additional signs of toxicity to the conceptus included treatment-related extra ribs and delayed ossification. Treatment was also associated with gross and skeletal malformations, such as median facial cleft, exencephaly, encephalocele, fused ribs, and anomalous vertebrae and exoccipitals. With the possible exception of the 5.0 mg/kg dose given on gestation day 8, nigericin doses associated with gross or skeletal malformations also resulted in observable maternal toxicity.     

Teratogenic effect of D-amphetamine sulphate: histodifferentiation and electrocardiogram pattern of mouse embryonic heart

Pregnant albino mice (ICR random-bred strain) received daily injections of 50 or 100 mg/kg body weight of D-amphetamine sulphate between days 9 and 11 of gestation. Parallel control animals were injected with saline solution. Treated mice were sacrificed on day 15 or 19 of gestation. The embryos were examined for gross malformations and direct embryonic ECG recordings were made; they were weighed, and their hearts were carefully dissected. Microscopic sections of the heart were stained with hematoxylin and eosin. It was found that high doses of D-amphetamine raised the incidence of mortality in the treated pregnant mice up to 40%. The resorption rate in the survivors was high (up to 58%) following the high dose of the drug. Up to 15% of the embryos from the treated groups showed gross malformations, including skeletal and eye malformations and exencephaly. The electrocardiogram (ECG) recordings in most 19-day-old embryos from both treated groups showed a pattern with prolonged Q-T interval (PQT), similar to that of control embryos in the intermediate developmental stages (days 14-16 of gestation). The ECG of control embryos (from day 17 on) resembled that of prenatal fetuses. Microscopically, the hearts of treated embryos showed a large number of undifferentiated cardiac myoblasts. It can be inferred that high doses of D-amphetamine affect embryonic development generally and delay the histodifferentiation of the myocardium, resulting in incomplete maturation of the cardiac muscles, thus leading to the immature ECG pattern, with PQT intervals.     

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.