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.     

Histological comparison of the effects of the splotch gene and retinoic acid on the closure of the mouse neural tube

The splotch gene (Sp) and all-trans retinoic acid (RA) interact to cause spina bifida in mouse embryos. To investigate the mechanisms of action of the two, the spinal regions of Sp homozygotes, RA-treated wild-type, and control wild-type embryos were examined histologically by light microscopy on day 9 of gestation. The mean numbers of cells per section in the neural tube, mesoderm, and notochord were determined, along with the percentages of mitotic and pyknotic nuclei and the numbers of migrating neural crest cells. As well, the effect of Sp and RA on the extracellular matrix was studied histochemically with Alcian blue staining for glycosaminoglycans. The main defect in Sp homozygotes was a marked reduction in the number of migrating neural crest cells and the amount of extracellular matrix around the neural tube. Retinoic acid, on the other hand, caused a number of disruptions in the embryo, including abnormalities in the position of the notochord and the shape of the neural tube. Sp and RA delay neural tube closure and thus cause neural tube defects, through different mechanisms. However, the combined effects of the gene and teratogen on the embryo lead to a greater inhibition of neural tube closure than when either is present separately.     

Early morphological abnormalities in splotch mouse embryos and predisposition to gene- and retinoic acid-induced neural tube defects

Genetic and environmental factors contribute to an individual's neural tube defect liability. In the mouse, the gene mutation Splotch (Sp) causes a pigmentation defect in heterozygotes while homozygotes have spina bifida +/- exencephaly. Splotch homozygotes, heterozygotes, and wild-type embryos were examined for somite number, anterior neuropore closure, and posterior neuropore length. The aim was to distinguish potentially affected homozygotes early in pathogenesis and find a morphological basis for increased teratogen susceptibility in heterozygotes. Posterior neuropore closure as well as anterior neuropore closure was significantly delayed in potentially affected Sp as compared to wild-type litter embryos exceeding the incidence found in day-10-diagnosed homozygotes. Part of this excess was attributed to a transient delay in heterozygotes which in turn might predispose to retinoic acid-induced neural tube defects. This idea was supported by an outcross of Sp heterozygote males by inbred SWV females and wild-type males by SWV where a significant increase in retinoic acid-induced neural tube defects was found in Sp carrier litters.     

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.     

Quantification and localization of expression of the retinoic acid receptor-beta and -gamma mRNA isoforms during neurulation in mouse embryos with or without spina bifida

BACKGROUND: Previous studies observed that retinoic acid receptor-gamma (RARgamma) is expressed in the open caudal neuroepithelium but that RARbeta is expressed in the closed neural tube. Furthermore, retinoic acid (RA) induces RARbeta expression, a molecular event associated with neural tube closure, but treatment with RA at the appropriate gestation time causes failure of neural tube closure. Since there are four isoforms of RARbeta, perhaps the isoforms expressed in the closed neural tube and induced by RA are different. To investigate the hypothesis that the switch from RARgamma to RARbeta is mechanistically linked to neural tube closure, this study determined the concentrations and distributions of RARbeta and RARgamma isoforms in mouse embryos with RA-induced neural tube defects and in splotch (Sp) mutant embryos with spina bifida. METHODS: Absolute concentrations of RARbeta and RARgamma isoforms were determined throughout primary neurulation (gestational day 8.5-10.0) in treated or untreated C57BL/6J mouse whole embryos by ribonuclease protection analysis. Treatment consisted of an oral dose of 100 mg/kg of all-trans-RA on gestational day 8.5. Spatial distributions of RARbeta and RARgamma were examined in RA-treated and Sp mutant embryos by in situ hybridization. RESULTS: RARbeta2, gamma1, and gamma2 were expressed in untreated embryos and were induced 4.5-, 1.6-, and 4.0-fold, respectively, 4 hr after treatment with RA. In embryos with RA-induced spina bifida, RARbeta2 was expressed in the closed neural tube while RARgamma1 and RARgamma2 were expressed in the open caudal neuroepithelium. In splotch mice with spina bifida, the boundary between RARbeta and RARgamma did not correspond to the site of neural tube closure. CONCLUSIONS: In RA-treated embryos, the relationship between RARbeta expression in the closed and RARgamma in the open caudal neuroepithelium was not altered. However, in splotch embryos with spina bifida, the juncture between RARbeta and RARgamma expression remained in the same anatomical position in the neuroepithelium irrespective of the neural tube closure status and suggests that the switch from RARgamma to RARbeta expression in the closing caudal neuroepithelium may not be causally linked to neural tube closure in the splotch mutant.     

Immunohistochemical localization of chondroitin and heparan sulfate proteoglycans in pre-spina bifida splotch mouse embryos.

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/ +) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction in the frequency of neural tube defects in splotch mice by retinoic acid

In the homozygous state, the splotch (Sp) gene causes spina bifida and exencephaly. Close to 25% of the embryos from Sp/ + X Sp/+ litters are affected. The frequency of these defects is significantly reduced by maternal treatment with 5 mg/kg retinoic acid on day 9 of gestation. There is no significant increase in the resorption frequency with this treatment, indicating that the fall in the frequency of neural tube defects is not due to differential mortality of the affected fetuses. The effects of retinoic acid are time specific, with treatment at different times on day 9 having the greatest influence on either the anterior or posterior neuropore. Treatment on day 8 with the same dose of retinoic acid causes an increase in both resorptions and neural tube defects, although only the increase in the former was significant.     

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.     

Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect

A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by 'cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. We used focal cell labelling and orthotopic grafting, followed by whole embryo culture, to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonisation of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resembled wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotch cardiac neural crest defect, we performed reciprocal grafts of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrated normally in the +/+ environment, and +/+ cells migrated normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurred when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2H environment. We conclude that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment.     

Prevention of spinal neural tube defects in the mouse embryo by growth retardation during neurulation

Homozygous mutant curly tail mouse embryos developing spinal neural tube defects (NTD) exhibit a cell-type-specific abnormality of cell proliferation that affects the gut endoderm and notochord but not the neuroepithelium. We suggested that spinal NTD in these embryos may result from the imbalance of cell proliferation rates between affected and unaffected cell types. In order to test this hypothesis, curly tail embryos were subjected to influences that retard growth in vivo and in vitro. The expectation was that growth of unaffected rapidly growing cell types would be reduced to a greater extent than affected slowly growing cell types, thus counteracting the genetically determined imbalance of cell proliferation rates and leading to normalization of spinal neurulation. Food deprivation of pregnant females for 48 h prior to the stage of posterior neuropore closure reduced the overall incidence of spinal NTD and almost completely prevented open spina bifida, the most severe form of spinal NTD in curly tail mice. Analysis of embryos earlier in gestation showed that growth retardation acts by reducing the incidence of delayed neuropore closure. Culture of embryos at 40.5 degrees C for 15-23 h from day 10 of gestation, like food deprivation in vivo, also produced growth retardation and led to normalization of posterior neuropore closure. Labelling of embryos in vitro with [3H]thymidine for 1 h at the end of the culture period showed that the labelling index is reduced to a greater extent in the neuroepithelium than in other cell types in growth-retarded embryos compared with controls cultured at 38 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Analysis of the effect of the loop-tail gene in mouse embryos]

Mitotic index and parameters of the cell cycle were determined in the brain and spinal cord of 10 days old Lp/Lp and +/+ mouse embryos. The mitotic index and duration of the cell cycle periods proved to be the same for embryos of both the genotypes. The generation time of the brain and spinal cord cells both in the mutant and normal embryos is 9 hrs, durations of S- and G2-periods 6 and 1, resp., and the total duration of G1- and M-periods 2. The gene Lp does not interact with the gene Sp in double heterozygotes. The gene Lp does not manifest itself in the cells of differentiating central nervous system and the failure of the neural tube closure is not due to the changes in the proliferative activity of its cells and is a secondary gene effect.     

Maternal treatment with teratogen causes congenital malformations in mouse embryos

Heterozygotes for the tail-short (Ts) mutant gene in the Balb/c strain have minor skeletal defects and a short, kinky tail. If heterozygote Ts/+ mothers are mated with normal-tail +/+ males and are treated with teratogenic doses of trypan blue on the eighth day of pregnancy, the mutant F1 heterozygotes develop exencephaly, folded neural tube and spina bifida significantly more often than non-mutants. This is indicative of gene-teratogen interaction, with the Ts gene increasing the embryo's susceptibility to trypan blue-induced neural tube defects.     

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.     

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.     

Impact of methylenetetrahydrofolate reductase deficiency and low dietary folate on the development of neural tube defects in splotch mice

BACKGROUND: The etiology of neural tube defects (NTDs) is multifactorial, with environmental and genetic determinants. Folate supplementation prevents the majority of NTDs, and a polymorphism in methylenetetrahydrofolate reductase (MTHFR) has become recognized as a genetic risk factor. The mechanisms by which folate affects NTD development are unclear. The Splotch (Sp) mouse is a well-characterized mouse model for studying spontaneous NTDs. To assess the potential interaction between folate metabolism and the Sp mutant in NTD development, we studied mice with both Sp and Mthfr mutations, as well as the interaction between Sp and low dietary folate. METHODS: Wild-type, single Mthfr+/-mutant, single Sp/+mutant, and double mutant (Mthfr+/-, Sp/+) female mice were mated with males of the same genotype. Embryos were examined for NTDs on gestational day (GD) 13.5. To investigate the effects of folate deficiency on Sp mice, Sp/+female mice were fed a control diet (CD), a moderately folic acid-deficient diet (MFADD), or a severely folic acid-deficient diet (SFADD). They were mated with Sp/+males and the embryos were examined. RESULTS: There were no differences in the incidence or severity of NTDs in embryos from double-mutant mating pairs compared to those from single Sp mutants. Embryos from Mthfr+/-dams did not exhibit NTDs. Diets deficient in folate did not influence the incidence or severity of NTDs in embryos from Sp/+mice. CONCLUSIONS: We did not observe an interaction between Sp and Mthfr mutations, or between the Sp mutation and low dietary folate, in NTD development in Splotch mice.     

Analysis of ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2 in human neural tube defects suggests a possible association with alleles in ALDH1A2

BACKGROUND: Vitamin A (retinol), in the form of retinoic acid (RA), is essential for normal development of the human embryo. Studies in the mouse and zebrafish have shown that retinol is metabolized in the developing spinal cord and must be maintained in a precise balance along the anteroposterior axis. Both excess and deficiency of RA can affect morphogenesis, including failures of neural tube closure. METHODS: We chose to investigate 5 genes involved in the metabolism or synthesis of RA, ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2, for their role in the development of human neural tube defects, such as spina bifida. RESULTS: An association analysis using both allelic and genotypic single-locus tests revealed a significant association between the risk for spina bifida and 3 polymorphisms in the gene ALDH1A2; however, we found no evidence of a significant multilocus association. CONCLUSIONS: These results may suggest that polymorphisms in ALDH1A2 may influence the risk for lumbosacral myelomeningocele in humans.     

Teratogenic effects of transplacental transfusion of heterologous antisera simulated in an experimental model using in vitro whole rat embryo culture

The effects of the transplacental transfusion of heterologous rabbit-anti-rat antiserum (RAR antiserum) and subsequent immunological interaction on the development of 9-10 days old rat embryos (stages 8-10 somites) were studied using an in vitro whole rat embryo culture. Transplacental transfusion was simulated by the embryonic intracardiac microinjection of approximately 0.5 microliter RAR antiserum, followed by an incubation period of 24 and 48 hours. All the tested embryos survived the incubation period. Embryos taken from the incubator after 24 hours showed signs of growth retardation and axial non-rotation, a delayed closure of the neural tube and ear vesicle, and a delayed formation of the foregut. They also had a moderate number of areas with local pathogenetic cell degeneration. Embryos taken from the incubator after 48 hours demonstrated signs of growth retardation and incomplete axial rotation. The formation of the foregut and closure of the neural tube was complete, with the exception of one embryo with a persisting open neuroporus posterior. All embryos displayed a considerable number of areas with local pathogenetic cell degeneration. The intracardiac injection technique is an elegant method to test the effects of teratogens administered directly into the embryonic circulation. The results demonstrate that heterologous antisera have teratogenic potential, believed to be due to an immunological reaction, with a particular sensitivity of the neurectoderm in 9-10 day old embryos.