Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.     

Ascorbic Acid Reverses Valproic Acid-Induced Inhibition of <Emphasis Type="Italic">Hoxa2</Emphasis> and Maintains Glutathione Homeostasis in Mouse Embryos in Culture

Valproic acid (VPA) has been shown to cause neural tube defects in humans and mice, but its mechanism of action has not been elucidated. We hypothesize that alterations in embryonic antioxidant status and Hoxa2 gene expression play an important role in VPA-induced teratogenesis. A whole embryo culture system was applied to explore the effects of VPA on total glutathione, on glutathione in its oxidized (GSSG) and reduced (GSH) forms [GSSG/GSH ratio] and on Hoxa2 expression in cultured CD-1 mouse embryos during their critical period of organogenesis. Our results show that VPA can (1) induce embryo malformations including neural tube defects, abnormal flexion, yolk sac circulation defects, somite defects, and craniofacial deformities such as fusion of the first and second arches, and (2) alter glutathione homeostasis of embryos through an increase in embryonic GSSG/GSH ratio and a decrease in total GSH content in embryos. Western blot analysis and quantitative real-time RT-PCR show that VPA can inhibit Hoxa2 expression in cultured embryos at both the protein and mRNA level, respectively. The presence of ascorbic acid in the culture media was effective in protecting embryos against oxidative stress induced by VPA and prevented VPA-induced inhibition of Hoxa2 gene expression. Hoxa2 null mutant embryos do not exhibit altered glutathione homeostasis, indicating that inhibition of Hoxa2 is downstream of VPA-induced oxidative stress. These results are first to suggest VPA may, in part, exert its teratogenicity through alteration of the embryonic antioxidant status and inhibition of Hoxa2 gene expression and that ascorbic acid can protect embryos from VPA-induced oxidative stress.     

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.     

Methionine and neural tube closure in cultured rat embryos: morphological and biochemical analyses

When headfold-stage rat embryos were cultured on cow serum, their neural tubes failed to close unless the serum was supplemented with methionine. Methionine deficiency did not appear to affect the ability of the neural epithelium to fuse as a type of fusion was observed between anterior and posterior regions of the open neural tube in methionine-deficient embryos. Although methionine deficiency reduced the cell density and mitotic indices of cranial mesenchyme and neural epithelial cells, this did not appear to be a factor in failure of the neural tube to close. For example, embryos cultured on diluted cow serum also had fewer mesenchymal cells yet could complete neural tube closure if provided with methionine. Examination of the tips of the neural folds suggested that microfilament contraction could be involved; in the absence of methionine the neural folds failed to turn in. This possibility was supported by the reductions in neurite extension of isolated neural tubes cultured without methionine and by the reductions in microfilament associated methylated amino acids contained in embryo neural tube proteins.     

Lack of attenuation of valproic acid-induced effects by folinic acid in rat embryos in vitro

The anticonvulsant drug valproic acid (VPA) is suspected to be a developmental toxicant in humans, inducing primarily neural tube defects. The mechanism for this effect is unknown, but it has been suggested that the drug may act via a deficiency of the vitamin folic acid. We examined this possibility by concurrent treatment of rat embryos in a whole embryo culture system with VPA and folinic acid (FA), a folic acid derivative. Groups of CD rat embryos were treated with various concentrations of VPA, various concentrations of FA, or a combination of a teratogenic dose of VPA plus various levels of FA. At the end of the 44 hour culture period, each embryo was evaluated for viability (presence of a heartbeat), yolk sac circulation, presence of any malformations, morphological score, crown-rump and head lengths, as well as DNA and protein contents. The anticonvulsant did not decrease viability but did decrease yolk sac circulation and all growth and developmental endpoints in a dose-responsive manner. There was also a dose-related increase in the incidence of open neural tubes. The addition of FA alone had no significant effect on growth and development. When various concentrations of FA were added simultaneously with a teratogenic dose of VPA, there was no decrease in the incidence of open neural tubes. Growth and developmental endpoints were altered in a somewhat random fashion but were never increased to the control level. The lack of attenuation by FA was not due to instability of the compound in the culture system, nor was there a difference in the amount of FA present in the exocoelomic fluid of VPA-treated and control embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Identification of early-responsive genes correlated to valproic acid-induced neural tube defects in mice

BACKGROUND: Valproic acid (VPA) causes the failure of neural tube closure in newborn mice. However, the molecular mechanism of its teratogenesis is unknown. This study was conducted to investigate the genomewide effects of VPA disruption of normal neural tube development in mice. METHODS: Microarray analysis was performed on the head part of NMRI mouse embryos treated for 1 hr with VPA on gestational day (GD) 8. Subsequently, we attempted to isolate genes that changed in correlation with the teratogenic action of VPA by employing reduced teratogenic VPA analogs, valpromide (VPD) and valnoctamide (VCD), in a real-time PCR study. RESULTS: Microarray results demonstrated that during neurulation, many genes, some of whose functions are known and some unknown, were either increased or decreased after VPA injection. Some genes were affected by VPD or VCD in the same way as VPA, but others were not changed by the analogs. In this way, our system identified 11 increased and 20 decreased genes. Annotation analysis revealed that the increased genes included gadd45b, ier5, per1, phfl3, pou3f1, and sox4, and the decreased genes included ccne2, ccnl, gas5, egr2, sirt1, and zfp105. CONCLUSIONS: These findings demonstrate that expression changes in genes having roles in the cell cycle and apoptosis pathways of neural tube cells were strongly expected to relate to the teratogenic, but not antiepileptic, activity of VPA. Our approach has allowed the expansion of the catalog of molecules immediately affected by VPA in the developing neural tube.     

SEM observations of the neural fold associated with neurulation in the rat

The topography of the ectoderm was examined by scanning electron microscopy during neurulation in rat embryos at stage 24 (somites 8-11). A zone of altered cell morphology was observed along the crest of neural folds. This zone was located between the presumptive neural tube and the surface ectoderm and exhibited numerous rounded cell blebs, immediately prior to fusion between the folds. It is suggested that the observed surface alterations may reflect a change in the properties of the altered cell which correlate with initial adhesion between the folds.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.     

Immunohistochemical localisation of chondroitin sulphate proteoglycans and the effects of chondroitinase ABC in 9- to 11-day rat embryos

Studies on cell behaviour in vitro have indicated that the chondroitin sulphate proteoglycan (CSPG) family of molecules can participate in the control of cell proliferation, differentiation and adhesion, but its morphogenetic functions had not been investigated in intact embryos. Chondroitin/chondroitin sulphates have been identified in rat embryos at low levels at the start of neurulation (day 9) and at much higher levels on day 10. In this study we have sought evidence for the morphogenetic functions of CSPGs in rat embryos during the period of neurulation and neural crest cell migration by a combination of two approaches: immunocytochemical localization of CSPG by means of an antibody, CS-56, to the chondroitin sulphate component of CSPG, and exposure of embryos to the enzyme chondroitinase ABC. Staining of the CS-56 epitope was poor at the beginning of cranial neurulation; bright staining was at first confined to the primary mesenchyme under the convex neural folds late on day 9. In day 10 embryos, all mesenchyme cells were stained, but at different levels of intensity, so that primary mesenchyme, neural crest and sclerotomal cells could be distinguished from each other. Basement membranes were also stained, particularly bright staining being present where two epithelial were basally apposed, e.g., neural/surface ectoderms, dorsal aorta/neural tube, prior to migration of a population of cells between them. Staining within the neural epithelium was first confined to the dorsolateral edge region, and associated with the onset of neural crest cell emigration; after neural tube closure, neuroepithelial staining was more general. Neural crest cells were stained during migration, but the reaction was absent in areas associated with migration end-points (trigeminal ganglion anlagen, frontonasal mesenchyme). Embryos exposed to chondroitinase ABC in culture showed no abnormalities until early day 10, when cranial neural crest cell emigration from the neural epithelium was inhibited and neural tube closure was retarded. Sclerotomal cells failed to take their normal pathway between the dorsal aorta and neural tube. Correlation of the results of these two methods suggests: (1) that by decreasing adhesiveness within the neural epithelium at specific stages, CSPG facilitates the emigration of neural crest cells and the migratory movement of neuroblasts, and may also provide increased flexibility during the generation of epithelial curvatures; (2) that by decreasing the adhesiveness of fibronectin-containing extracellular matrices, CSPG facilitates the migration of neural crest and sclerotomal cells. This second function is particularly important when migrating cells take pathways between previously apposed tissues.     

Integrity of the methylation cycle is essential for mammalian neural tube closure

BACKGROUND: Closure of the cranial neural tube during embryogenesis is a crucial process in development of the brain. Failure of this event results in the severe neural tube defect (NTD) exencephaly, the developmental forerunner of anencephaly. METHODS: The requirement for methylation cycle function in cranial neural tube closure was tested by treatment of cultured mouse embryos with cycloleucine or ethionine, inhibitors of methionine adenosyl transferase. Embryonic phenotypes were investigated by histological analysis, and immunostaining was performed for markers of proliferation and apoptosis. Methylation cycle intermediates s-adenosylmethionine and s-adenosylhomocysteine were also quantitated by tandem mass spectrometry. RESULTS: Ethionine and cycloleucine treatments significantly reduced the ratio of abundance of s-adenosylmethionine to s-adenosylhomocysteine and are, therefore, predicted to suppress the methylation cycle. Exposure to these inhibitors during the period of cranial neurulation caused a high incidence of exencephaly, in the absence of generalized toxicity, growth retardation, or developmental delay. Reduced neuroepithelial thickness and reduced density of cranial mesenchyme were detected in ethionine-treated but not cycloleucine-treated embryos that developed exencephaly. Reduced mesenchymal density is a potential cause of ethionine-induced exencephaly, although we could not detect a causative alteration in proliferation or apoptosis prior to failure of neural tube closure. CONCLUSIONS: Adequate functioning of the methylation cycle is essential for cranial neural tube closure in the mouse, suggesting that suppression of the methylation cycle could also increase the risk of human NTDs. We hypothesize that inhibition of the methylation cycle causes NTDs due to disruption of crucial reactions involving methylation of DNA, proteins or other biomolecules.     

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.     

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

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

Morphological differences elicited by two weak acids, retinoic and valproic, in rat embryos grown in vitro

We compared in rat whole-embryo culture the morphological changes elicited by valproic acid (VPA) with those elicited by trans-retinoic acid (RA). Rat embryos explanted on day 9.5 of gestation were treated on day 10 with RA or VPA at concentrations producing equivalent reductions in embryonic protein. The concentrations selected for morphological assessment by scanning and transmission electron microscopy, 2.3 and 800 microM, respectively, for RA and VPA, produced approximately a 50% incidence of abnormally open anterior neuropores in initial range-finding experiments in the culture system. Protein and DNA analyses were also performed on corresponding groups of embryos at three different doses. With concurrent control groups used as reference standards, the two treatment groups were compared for differences in external and internal morphology, protein and DNA contents, and growth indices. While certain variables responded similarly in the two treatment groups, e.g., the growth variables, protein and DNA contents, each drug produced selective morphological effects. Whereas treatment with RA produced underdeveloped branchial arches, symmetrically cleft cranial defects resulting in openings in rhombencephalic and prosencephalic regions, and exteriorized neural tissue in the caudal neuropore region, VPA produced irregular clefts with wavy margins along the entire length of the neural tube, and an open caudal neuropore without eversion of the neuroepithelium, while producing no detectable effect on the branchial arches. The similar effects of these two drugs on protein and DNA contents suggest comparable degrees of overall toxicity; however, the dissimilar effects on neural tube and branchial arches, coupled with the large difference in concentration of the drug required to produce the effects, add to the evidence that their mechanisms for elicitation of abnormal development are qualitatively different.     

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.     

Neurulation continues: The parade commander is… apical constriction

Neurulation is traditionally defined as the process of closure of the neural tube. New data have shown that the major driving forces of neurulation continue to operate with the closure of the neural tube, at least until the central canal of the neural tube has formed. Owing to this, the paper proposes to distinguish two periods of neurulation. According to these notions, early neurulation corresponds to the period of closure of the neural tube, and late neurulation corresponds to the period of formation of the central canal. Examples of neural tube defects that affect late neurulation are discussed.     

Tissue, cellular and sub-cellular localization of the Vangl2 protein during embryonic development: effect of the Lp mutation

Loop-tail (Lp) mice show a very severe neural tube defect, craniorachischisis, which is caused by mis-sense mutations in the Vangl2 gene. The membrane protein Vangl2 belongs to a highly conserved group of proteins that regulate planar polarity in certain epithelia, and that are also important for convergent extension movements during gastrulation and neurulation. A specific anti-Vangl2 antiserum was produced and used to examine the tissue, cell type, and sub-cellular localization of Vangl2 during embryogenesis. Vangl2 protein is expressed at high levels in the neural tube and shows a dynamic expression profile during neurulation. After neural tube closure, robust Vangl2 staining is detected in several neural and neurosensory tissues, including cerebral cortex, dorsal root ganglia, olfactory epithelium, retina, mechanosensory hair cells of the cochlea, and optic nerve. Vangl2 is also expressed during organogenesis in a number of tubular epithelia, including the bronchial tree, intestinal crypt/villus axis, and renal tubular segments derived from ureteric bud and from metanephric mesenchyme. Examination of Vangl2 localization in the neural tubes and cochleas of the normal and Lp/Lp embryos shows disruption of normal membrane localization of Vangl2 in independent alleles at Lp (Lp, Lp(m1Jus)) as well as overall decrease in the expression level.