Accumulation of basement membrane-associated hyaluronate is reduced in the posterior neuropore region of mutant (curly tail) mouse embryos developing spinal neural tube defects

We investigated the accumulation of newly synthesized glycoconjugates during spinal neurulation in mutant curly tail mouse embryos, a proportion of which develop lower spinal neural-tube defects (NTD). Embryos undergoing closure of the posterior neuropore (27- to 29-somite stage) were labeled in vitro with [3H]glucosamine, and [3H]glycoconjugates were analyzed by ion-exchange chromatography. Mutant embryos undergoing normal spinal neurulation exhibited a pattern of glycoconjugate accumulation closely similar to that observed for nonmutant embryos (Copp and Bernfield, 1988, Dev. Biol. 130, 573-582). Mutant embryos developing spinal NTD accumulated reduced amounts of [3H]hyaluronate specifically in the posterior neuropore region. Other embryonic regions and other glycoconjugates appeared unaffected by the developmental abnormality. Autoradiographic analysis of labeled curly tail embryos confirmed that [3H]hyaluronate accumulates in reduced amounts in the posterior neuropore region and indicated that this reduction is mainly localized to the site of developing basement membranes, beneath the neuroepithelium and around the notochord. Accumulation of [3H]hyaluronate in the interstitial mesenchymal matrix of the posterior neuropore region is not consistently affected in embryos developing spinal NTD. These results provide support for a role for basement-membrane hyaluronate in lower spinal neurulation.     

A cell-type-specific abnormality of cell proliferation in mutant (curly tail) mouse embryos developing spinal neural tube defects

The mouse mutant curly tail (ct) provides a model system for studies of neurulation mechanisms. 60% of ct/ct embryos develop spinal neural tube defects (NTD) as a result of delayed neurulation at the posterior neuropore whereas the remaining 40% of embryos develop normally. In order to investigate the role of cell proliferation during mouse neurulation, cell cycle parameters were studied in curly tail embryos developing spinal NTD and in their normally developing litter-mates. Measurements were made of mitotic index, median length of S-phase and percent reduction of labelling index during a [3H]thymidine pulse-chase experiment. These independent measures of cell proliferation rate indicate a reduced rate of proliferation of gut endoderm and notochord cells in the neuropore region of embryos developing spinal NTD compared with normally developing controls. The incidence of cell death and the relative frequency of mitotic spindle orientations does not differ consistently between normal and abnormal embryos. These results suggest a mechanism of spinal NTD pathogenesis in curly tail embryos based on failure of normal cell proliferation in gut endoderm and notochord.     

Curvature of the caudal region is responsible for failure of neural tube closure in the curly tail (ct) mouse embryo.

Delayed closure of the posterior neuropore (PNP) occurs to a variable extent in homozygous mutant curly tail (ct) mouse embryos, and results in the development of spinal neural tube defects (NTD) in 60% of embryos. Previous studies have suggested that curvature of the body axis may delay neural tube closure in the cranial region of the mouse embryo. In order to investigate the relationship between curvature and delayed PNP closure, we measured the extent of ventral curvature of the neuropore region in ct/ct embryos with normal or delayed PNP closure. The results show significantly greater curvature in ct/ct embryos with delayed PNP closure in vivo than in their normal littermates. Reopening of the posterior neuropore in non-mutant mouse embryos, to delay neuropore closure experimentally, did not increase ventral curvature, suggesting that increased curvature in ct/ct embryos is not likely to be a secondary effect of delayed PNP closure. Experimental prevention of ventral curvature in ct/ct embryos, brought about by implantation of an eyelash tip longitudinally into the hindgut lumen, ameliorated the delay in PNP closure. We propose, therefore, that increased ventral curvature of the neuropore region of ct/ct embryos imposes a mechanical stress, which opposes neurulation and thus delays closure of the PNP. Increased ventral curvature may arise as a result of a cell proliferation imbalance, which we demonstrated previously in affected ct/ct embryos.     

Abnormalities of neural tube formation in pre-spina bifida splotch-delayed mouse embryos

The splotch-delayed homozygous mutant (Spd/Spd) develops spina bifida with or without exencephaly, has spinal ganglia abnormalities, and delays in posterior neuropore closure and neural crest cell emigration. The heterozygote (Spd/+) has a pigmentation defect, and occasionally neural tube defects. To investigate the underlying mechanisms, we compared the neuroepithelium in the posterior neuropore region of cytogenetically identified 15-18 somite pair Spd/Spd, Spd/+, and +/+ mouse embryos by transmission electron and light microscopy. The notochordal area and cell number in the non-fused neuroepithelium region of Spd/Spd and Spd/+ embryos were significantly reduced compared to those of normal (+/+) embryos, which suggests an abnormality in notochord elongation. In the mesoderm, the mean cell number and mean ratio of cell number to area in the non-fused region were significantly lower in the Spd/Spd compared with +/+ embryos. The distance of exposed neuroepithelium above the mesoderm in the just-fused region was significantly lower in the Spd/Spd versus +/+ embryos, which may indicate an insufficient force exerted by the mesoderm during neural tube closure. Within the neuroepithelium, significantly more intercellular space was found in Spd/Spd than in +/+ embryos indicating disorganization. The basal lamina was poorly organized and the formation delayed around the neural tube in Spd/Spd and Spd/+ embryos. All together, these results suggest an early abnormality in interactions among the neuroepithelium, mesoderm, and notochord, which may lead to the delay or inhibition of neural tube closure observed in Spd/Spd mutants.     

Retinoic acid-induced spina bifida: evidence for a pathogenetic mechanism

Treatment of C57Bl/6J mice with three successive doses of all-trans retinoic acid (28 mg kg-1 body weight) on 8 day, 6 h (8d,6h), 8d,12h, and 8d,18h of gestation resulted in a high incidence (79%, 31/39 fetuses) of spina bifida with myeloschisis (spina bifida aperta) in near term fetuses. Twelve hours following the last maternal dose (9d,6h), the caudal aspects of treated embryos, were abnormal, with eversion of the neural plate at the posterior neuropore, as compared to its normal concavity in comparably staged control specimens. This eversion persisted in affected embryos through the time that the posterior neuropore should normally close. The distribution of cell death in control and experimental embryos was determined using vital staining with Nile blue sulphate and with routine histological techniques. Twelve hours following the maternal dosing regimen, experimental embryos showed evidence of excessive cell death, predominantly in the mesenchyme associated with the primitive streak and in the endoderm of the tail gut, both of which are readily identifiable sites of physiological cell death at this stage of development. In addition, the presumptive trunk neural crest cells located in the dorsal midline, cranial to the posterior neuropore, exhibited a marked amount of cell death in the experimental embryos. We propose that the major factor in the generation of spina bifida in this model is excessive cell death in the tail gut and mesenchyme ventral to the neuroepithelium of the posterior neuropore.(ABSTRACT TRUNCATED AT 250 WORDS)     

The induction of tail malformations in trisomy 16 mouse fetuses heterozygous for the curly tail recessive gene

The mouse mutant curly tail is thought to be inherited as an autosomal recessive (ct/ct) with incomplete penetrance so that approximately 60% of ct/ct individuals exhibit the curly tail (CT) phenotype. By outcrossing ct/ct with mouse stock carrying specific heterozygous combinations of Robertsonian (Rb) chromosomes, trisomy 16 (Ts16) and Ts19 mouse fetuses (and their chromosomally balanced littermates) were derived which were heterozygous for the ct gene. All of the Ts16 (ct/Rb;Rb) fetuses, studied between days 14-19 gestation had tail malformations, 86% of which were tail flexion defects (TFD) apparently very similar to the curly tail phenotype. Neither Ts19 nor any of the chromosomally balanced (ct/Rb) littermates from both experimental crosses showed any type of tail or other spinal malformation. At the 27-29 somite stage of development, Ts16 (ct/Rb;Rb) fetuses did not show any significant delay in the closure of the posterior neuropore (PNP) compared with their littermate controls, suggesting that the tail malformation observed in Ts16 (ct/Rb;Rb) occur as a result of mechanisms which differ significantly from those thought to be responsible to causing the curly tail malformation.     

Morphometric analysis of the forebrain anomalies in the delayed Splotch mutant embryo

The early development of delayed Splotch mouse embryos was examined histologically using scanning electron microscopy and morphometric techniques. Embryos obtained from matings of mice heterozygous for the delayed Splotch gene exhibited a high incidence of lumbosacral (25%) or cephalic (7%) neural tube defects. The lumbosacral neural tube defects extended from the posterior neuropore region to the tip of the tailbud; cephalic neural tube closure defects were found in the hindbrain and midbrain regions. The frontal region of affected embryos was abnormal in that it was reduced in size, particularly in the developing midface. Histologically, the forebrain region of affected embryos appeared reduced, and the luminal surface of the neuroepithelium was often irregular and infolded. To quantify these alterations and to determine their contribution to the final form of the region, size and areal measurements were recorded and served as input for principal component and cluster analytic techniques. In affected embryos, significant reductions were found in lumen size, in neuroepithelial area but not thickness, and in overall area of forebrain but not hindbrain. Principal component analysis of data from unaffected embryos produced two factors, one containing hindbrain variables and the second forebrain variables; for the affected embryos, three factors were extracted. The first loaded on variables that measured the thickness and area of the neuroepithelium, the second on forebrain variables, and the third on hindbrain variables. Factor scores were then generated from a pooled analysis of normal and affected cases and were analyzed using cluster analysis. Three clusters were identified: one contained eight affected embryos with cephalic neural tube defects; another contained nine affected embryos with lumbosacral neural tube defects and five normal embryos; and the final cluster contained ten unaffected embryos. These results suggest a major role of the delayed Splotch gene on the neuroepithelium itself and support the suggested role of cerebrospinal fluid pressure in normal forebrain histogenesis.     

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).     

Glycosaminoglycans vary in accumulation along the neuraxis during spinal neurulation in the mouse embryo

We have utilized the method of whole embryo culture for metabolic labeling of mouse embryos with [3H]glucosamine during closure of neural folds at the posterior neuropore (27- to 29-somite stage). Accumulations of newly synthesized glycopeptides, lactosaminoglycans, hyaluronate, and sulfated glycosaminoglycans (GAG) were assessed by ion-exchange chromatography of glycoconjugates isolated from labeled embryos. Accumulation of hyaluronate and sulfated GAG was greatest in the posterior neuropore and decreased progressively toward the hindbrain where neurulation was already complete. Hyaluronate comprised a progressively smaller proportion of total newly synthesized glycoconjugate from the posterior neuropore toward the cranial region and glycopeptides showed the opposite trend. Sulfated GAG and lactosaminoglycans showed no consistent differences in relative abundance along the neuraxis. Autoradiographic analysis of newly synthesized glycoconjugates revealed especially heavy incorporation into developing basement membranes, beneath the neuroepithelium and around the notochord, in the posterior neuropore and recently closed neural tube regions, but not at more cranial levels of the neuraxis. Predigestion of sections with a specific hyaluronidase showed a significant quantity of this glycoconjugate to be hyaluronate. These results are consistent with a role for neuroepithelial and notochordal basement membrane hyaluronate in spinal neurulation.     

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.     

Effects of folic acid and homocysteine on spinal cord morphology of the chicken embryo

Maternal ingestion of folic acid (FA) reduces neural tube defects, which are associated with high homocysteine levels. Present study evaluated the effects of FA and homocysteine on cell proliferation and cell adhesion, as well as on apoptosis, throughout the development of the spinal cord and mesenchyme of chicken embryos. Normal closure of the neural tube and a regular distribution of the mesenchymal cells were observed in control and FA-treated embryos. All homocysteine-treated embryos and also 6 of 10 embryos treated with FA + homocysteine showed failure of closure of the neural tube. Homocysteine decreased the thickness of the mantle and marginal layers of the spinal cord, and FA did not prevent this effect. FA treatment reversed the decrease of proliferating cells in the spinal cord induced by homocysteine. FA-treated embryos showed the highest numerical density of apoptotic cells. Homocysteine treatment reduced NCAM expression in both spinal cord and mesenchymal tissue, and FA prevents this effect. These results are important because they demonstrate in situ that the imbalance between FA and homocysteine levels can lead to disruptions in spinal cord development, changing proliferation, apoptosis, and cell adhesion and consequently changing the arrangement of the spinal cord layers.     

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.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.     

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.     

Phenytoin embryotoxicity: role of enzymatic bioactivation in a murine embryo culture model

A murine embryo culture model was developed to study the potential contribution of enzymatic bioactivation to the teratogenicity of phenytoin. To assess the relative embryonic and maternal contributions to bioactivation, embryos were cultured respectively alone or in the presence of an exogenous source of cytochromes P-450 (P-450), which are thought to bioactivate phenytoin to a teratogenic reactive intermediate. Embryological development from gestational day 9 to day 10 was assessed, and bioactivation was quantified by the irreversible binding of radiolabeled phenytoin to embryonic protein. Embryos cultured with phenytoin and an exogenous P-450 bioactivating system showed a significant decrease in the incidence of turning and closure of the anterior neuropore, yolk sac diameter, and protein content as well as growth retardation. In the absence of an exogenous P-450 system, phenytoin did not decrease the incidence of turning or anterior neuropore closure but did cause growth retardation and a lesser but significant reduction in yolk sac diameter and embryonic protein content. An exogenous P-450 system enhanced the bioactivation of phenytoin, although significant activity also was detectable in embryos cultured without an exogenous bioactivating system. These results suggest that the embryo itself can enzymatically bioactivate embryotoxically significant amounts of phenytoin, and that bioactivation and embryotoxicity can be further enhanced, qualitatively and quantitatively, by an exogenous P-450 system, implicating a possible maternal contribution to phenytoin teratogenicity.