Neural Tube Closure Defects Research Articles

PCP effector gene Inturned is an important regulator of cilia formation and embryonic development in mammals

The PCP effector gene Inturned regulates planar cell polarity (PCP) and wing hair formation in Drosophila wings. In order to understand the roles for Inturned in mammalian embryonic development, we generated a null mutant allele for the mouse homologue of Inturned (Intu) via gene-targeting in ES cells. Mouse Intu null mutants are homozygous lethal at midgestation, exhibiting multiple defects including neural tube closure defects, abnormal dorsal/ventral patterning of the central nervous system and abnormal anterior–posterior patterning of the limbs resulting in severe polydactyly (7–9 digits each limb). The developmental processes affected by the Intu mutation are under the control of Hh signaling through Gli-family transcription factors. We found that in Intu mutant embryos the expression of Gli1 and Ptch1, two direct transcriptional targets of Hh signaling, is down-regulated, and the proteolytic processing of Gli3 is compromised. We further demonstrate that Intu plays significant roles in the formation of primary cilia both during embryonic development and in cultured fibroblasts. Finally, a cytoplasmic GFP-Intu fusion protein efficiently rescues the ciliogenic defects in Intu mutant cells. In conclusion, we show that PCP effector gene Intu is an important regulator of cilia formation, Hh signal transduction, and embryonic development in mammals.

Folic acid and soybean isoflavone combined supplementation protects the post-neural tube closure defects of rodents induced by cyclophosphamide in vivo and in vitro

To evaluate the neuroprotective effect of folic acid (FA) and soybean isoflavone (SIF) combined supplementation on the post-neural tube closure of rodents induced by cyclophosphamide (CPA) in vitro and in vivo, pregnant rats were randomly divided into control, model, solo-FA intervention, solo-SIF intervention and co-intervention of FA and SIF groups. Rats in solo-intervention groups and co-intervention group were treated with FA 0.7 mg/kg, SIF 160 mg/kg and FA 0.7 mg/kg + SIF 160 mg/kg at the duration of pregnancy, respectively. On the 13th day of gestation, control rats were given physiological saline and the other four groups were treated with CPA12.5 mg/kg. On the 14th day of gestation, three rats selected randomly from every group were executed and the ultrastructure, DNA damage and the proteins expressions of Bcl-2, Bax and P53 on embryo brains were checked. The rest of the rats were executed on the 20th day, the frequency of neural tube closure defects and fetus’ development indices were measured. In addition, T-SOD, MDA and NO in serum of the pregnant rats were checked. In vitro, the effect of FA and genistein on the apoptosis was determined. Compared with the model group, the malformation incidence was lower but fetus’ development indices were higher in the combination treated group. The combination decreased the damage of CPA, such as damaged nuclear DNA, early apoptotic morphological changes, Bax and P53 expressions on embryo brains and in vivo. Furthermore, T-SOD activity in serum of the pregnant rats increased and the levels of MDA and NO decreased in the combined supplementation group. Our study indicates the neuroprotection of FA and SIF combined administration is superior to solo treatment. Decrease of DNA damage and neuron apoptosis involved in the mechanisms. Furthermore, the up-regulation of Bcl-2 and the down-regulation of Bax and P53 proteins also participate in the effect.

Identification and characterization of a mouse recessive lethal mutation which displays cranial neural tube closure defects

Identification and characterization of a mouse recessive lethal mutation which displays cranial neural tube closure defects

Genetics and development of neural tube defects.

Congenital defects of neural tube closure (neural tube defects; NTDs) are among the commonest and most severe disorders of the fetus and newborn. Disturbance of any of the sequential events of embryonic neurulation produce NTDs, with the phenotype (eg anencephaly, spina bifida) varying depending on the region of neural tube that remains open. While mutation of > 200 genes is known to cause NTDs in mice, the pattern of occurrence in humans suggests a multifactorial polygenic or oligogenic aetiology. This emphasizes the importance of gene-gene and gene-environment interactions in the origins of these defects. A number of cell biological functions are essential for neural tube closure, with defects of the cytoskeleton, cell cycle and molecular regulation of cell viability prominent among the mouse NTD mutants. Many transcriptional regulators and proteins that affect chromatin structure are also required for neural tube closure, although the downstream molecular pathways regulated by these proteins is unknown. Some key signalling pathways for NTDs have been identified: over-activation of sonic hedgehog signalling and loss of function in the planar cell polarity (non-canonical Wnt) pathway are potent causes of NTD, with requirements also for retinoid and inositol signalling. Folic acid supplementation is an effective method for primary prevention of a proportion of NTDs in both humans and mice, although the embryonic mechanism of folate action remains unclear. Folic acid-resistant cases can be prevented by inositol supplementation in mice, raising the possibility that this could lead to an additional preventive strategy for human NTDs in future.

Target Deletion of the Cytoskeleton-Associated Protein Palladin Does Not Impair Neurite Outgrowth in Mice

Palladin is an actin cytoskeleton–associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90–92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice.

Regulation of cell surface protease matriptase by HAI2 is essential for placental development, neural tube closure and embryonic survival in mice

Hypomorphic mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis, including organ and digit duplications, atresia, fistulas, hypertelorism, cleft palate and hamartoma. SPINT2 encodes the transmembrane serine protease inhibitor HAI2 (placental bikunin), and the severe developmental effects of decreased HAI2 activity can be hypothesized to be a consequence of excess pericellular proteolytic activity. Indeed, we show here that HAI2 is a potent regulator of protease-guided cellular responses, including motogenic activity and transepithelial resistance of epithelial monolayers. Furthermore, we show that inhibition of the transmembrane serine protease matriptase (encoded by St14) is an essential function of HAI2 during tissue morphogenesis. Genetic inactivation of the mouse Spint2 gene led to defects in neural tube closure, abnormal placental labyrinth development associated with loss of epithelial cell polarity, and embryonic demise. Developmental defects observed in HAI2-deficient mice were caused by unregulated matriptase activity, as both placental development and embryonic survival in HAI2-deficient embryos were completely restored by the simultaneous genetic inactivation of matriptase. However, neural tube defects were detected in HAI2-deficient mice even in the absence of matriptase, although at lower frequency, indicating that the inhibition of additional serine protease(s) by HAI2 is required to complete neural development. Finally, by genetic complementation analysis, we uncovered a unique and complex functional interaction between HAI2 and the related HAI1 in the regulation of matriptase activity during development. This study indicates that unregulated matriptase-dependent cell surface proteolysis can cause a diverse array of abnormalities in mammalian development.

New Roles for Copper Metabolism in Cell Proliferation, Signaling, and Disease

Over the past 2 decades, the molecular mechanisms by which cells acquire, distribute, and utilize copper have been under intense investigation. Significant progress has been made in the identification of genes encoding copper homeostasis proteins and in fundamental aspects of their structure, function, and mechanisms of action in copper balance. A number of more comprehensive reviews of the field with respect to the genetics, structure, function, and physiology of copper metabolism have recently appeared elsewhere (1–4). Here, we review general mechanisms for eukaryotic copper metabolism at the cellular level in the context of recent discoveries in the field, identifying potential new functions for copper and copper metabolism proteins in cell signaling, gene expression, tumor cell metastasis, and resistance to anti-neoplastic drugs.

The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure

In the nematode Caenorhabditis elegans, the let-7 microRNA (miRNA) controls the timing of key developmental events and terminal differentiation in part by directly regulating lin-41. C. elegans lin-41 mutants display precocious cell cycle exit and terminal differentiation of epidermal skin cells. lin-41 orthologues are found in more complex organisms including both mice and humans, but their roles are not known. We generated Mlin41 mouse mutants to ascertain a functional role for Mlin41. Strong loss of function Mlin41 gene-trap mutants demonstrated a striking neural tube closure defect during development, and embryonic lethality. Like C. elegans lin-41, Mlin41 also appears to be regulated by the let-7 and mir-125 miRNAs. Since Mlin41 is required for neural tube closure and survival it points to human lin-41 (HLIN41/TRIM71) as a potential human development and disease gene.

Neogenin and RGMa Control Neural Tube Closure and Neuroepithelial Morphology by Regulating Cell Polarity

In humans, neural tube closure defects occur in 1:1000 pregnancies. The design of new strategies for the prevention of such common defects would benefit from an improved understanding of the molecular events underlying neurulation. Neural fold elevation is a key morphological process that acts during neurulation to drive neural tube closure. However, to date, the molecular pathways underpinning neural fold elevation have not been elucidated. Here, we use morpholino knock-down technology to demonstrate that Repulsive Guidance Molecule (RGMa)-Neogenin interactions are essential for effective neural fold elevation during Xenopus neurulation and that loss of these molecules results in disrupted neural tube closure. We demonstrate that Neogenin and RGMa are required for establishing the morphology of deep layer cells in the neural plate throughout neurulation. We also show that loss of Neogenin severely disrupts the microtubule network within the deep layer cells suggesting that Neogenin-dependent microtubule organization within the deep cells is essential for radial intercalation with the overlying superficial cell layer, thereby driving neural fold elevation. In addition, we show that sustained Neogenin activity is also necessary for the establishment of the apicobasally polarized pseudostratified neuroepithelium of the neural tube. Therefore, our study identifies a novel signaling pathway essential for radial intercalation and epithelialization during neural fold elevation and neural tube morphogenesis.

Phosphorylation of Gli2 by protein kinase A is required for Gli2 processing and degradation and the Sonic Hedgehog-regulated mouse development

In mice, Gli2 and Gli3 are the transcription factors that mediate the initial Hedgehog (Hh) signaling. In the absence of Hh signaling, the majority of the full-length Gli3 protein undergoes proteolytic processing into a repressor, while only a small fraction of the full-length Gli2 protein is processed. Gli3 processing is dependent on phosphorylation of the first four of the six protein kinase A (PKA) sites at its C-terminus. However, whether the same phosphorylation of Gli2 by PKA is required for Gli2 processing and, if so, whether such phosphorylation regulates additional Gli2 function are unknown. To address these questions, we mutated these PKA sites in the mouse Gli2 locus to create the Gli2P1-4 allele. Gli2P1-4 homozygous embryos die in utero and exhibit exencephaly, defects in neural tube closure, enlarged craniofacial structures, and an extra anterior digit. Analysis of spinal cord patterning shows that domains of motoneurons and V2, V1, and V0 interneurons are expanded to different degrees in both Gli2P1-4 single and Gli2P1-4;Shh double mutants. Furthermore, Gli2P1-4 expression prevents massive cell death and promotes cell proliferation in Shh mutant. Analysis of Gli2P1-4 protein in vivo reveals that the mutant protein is not processed and is twice as stable as wild type Gli2 protein. We also show that the Gli2 repressor can effectively antagonize Gli2P1-4 activity. Together, these results indicate that phosphorylation of Gli2 by PKA induces Gli2 processing and destabilization in vivo and plays an important role in the Hh-regulated mouse embryonic patterning.

Effect of sodium phenytoin concentration on neural tube development in the early stages of chicken embryo development

Animal and human research has shown that anticonvulsants are teratogens and pose a risk of causing fetal malformations. In various studies, the teratogenic effects of sodium phenytoin (PTH) in several systems have been investigated. Toe and finger, renal, and even facial malformations have been described in the literature. However, there is debate about whether the true risk of teratogenesis is lower or higher than previously reported for PTH. There is also little published information on the effect of this agent on neural tube closure in an embryological model. In this study, 0.1 mL of three different concentrations of PTH solution (mg/mL: 1, 3, 5) or vehicle was applied under the embryonic disc of specific pathogen-free Leghorn chicken embryos after 24 hours’ incubation. Incubation was continued until 72 hours of maturation. At 72 hours, all embryos were evaluated macroscopically and microscopically. There were serious neural tube closure defects in the embryos administered large amounts (0.5 mg) of PTH, but doses of 0.1 mg (subtherapeutic concentration for humans) and 0.3 mg (therapeutic concentration for humans) produced no statistically significant defects ( p = 0.05). The difference between the defects in the high concentration group and the other three groups was statistically significant. In our study PTH administered in a strict concentration regimen produced a lower level of neural tube closure-related defects than previously reported.

LUZP deficiency affects neural tube closure during brain development

LUZP is a leucine zipper-containing protein predominantly expressed in the brain. The functional significance of LUZP remains unknown. To explore the role of LUZP in brain development, a knockout mouse strain with a lacZ knock-in (Luzp-KO/lacZ-KI) has been established. LacZ reporter expression driven by the endogenous Luzp promoter was detected in the neuroepithelium and the cardiac tissue. Luzp−/− mice exhibited perinatal death, presumably due to the accompanied complex cardiovascular defects. Luzp−/− embryos displayed a cranial neural tube closure defect (NTD), with exposed brain tissues. Ectopic expression of Sonic-hedgehog, which is a protein known to be involved in neural tube closure, and elevated apoptosis were observed in the dorsal lateral neuroepithelium of the NTD Luzp−/− hindbrain. These findings assign a novel function of LUZP in the embryonic development of brain.

Influences of Reduced Expression of Maternal Bone Morphogenetic Protein 2 on Mouse Embryonic Development

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. In the course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3′ untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal mouse tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/–) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. The number of embryos exhibiting these abnormalities was increased when, due to different genotypes, expression levels of Bmp2 in maternal tissues were lower. These results suggest that the expression levels of Bmp2 in both embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds.

Ependymoblastomatous Exencephaly: A Unique Fetal Malformation

Exencephaly/anencephaly is a rare neural tube defect occurring early in embryogenesis. We report a 14-week-old fetus with exencephaly in whom central nervous system tissue was developed and preserved. There were 2 symmetrical structures grossly resembling cerebral hemispheres, which on histologic and ultrastructural study, consisted of a combination of ependymoblastomatous rosettes and canals and primitive neural tissue. The brainstem and spinal cord were partially normally formed, although descending tracts were not apparent. No cerebellar tissue was found. The eyes were formed. This appears to represent a rare example of exencephaly not covered by skin, which did not undergo necrosis and early transformation into a residual area cerebrovasculosa, characteristic of anencephaly. It may be appropriate to regard this as a unique neural tube closure defect that might be termed "ependymoblastomatous exencephaly."

Genetic and physical interaction between the NPHP5 and NPHP6 gene products.

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.

Prévention par l’acide folique des défauts de fermeture du tube neural : la question n’est toujours pas réglée

Between 1981 and 1996, several interventional studies proved the efficacy of periconceptional folic acid supplementation in the prevention of neural tube closure defects (NTCD), first in women at risk (with a previous case of NTCD) and also in women of the general population in age to become pregnant. The poor observance of this supplementation led several countries (USA, Canada, Chile...) to decide mandatory folic acid fortification of cereals, which permitted a 30% (USA) to 46% (Canada) reduction in the incidence of NTCD. Moreover, this benefit was accompanied by a diminished incidence of several other malformations and of stroke and coronary accidents in elderly people. However, several papers drew attention to an increased risk of colorectal and breast cancer in relation with high blood folate levels and the use of folic acid supplements. A controlled interventional study showed a higher rate of recurrence of colic adenomas and a higher percentage of advanced adenomas in subjects receiving 1mg/day of folic acid. A recent study demonstrated an abrupt reversal of the downward trend in colorectal cancer 1 year after the beginning of cereal folic acid fortification in the USA and Canada. Two studies also reported impaired cognitive functions in elder persons with defective vitamin B(12) status. Taken in aggregate, these studies question the wisdom of a nationwide, mandatory, folic acid fortification of cereals. As of today, despite their limited preventive efficacy, a safe approach is to keep our current French recommendations and to increase the awareness of all caregivers, so as to improve the observance of these recommendations.

Regulation of apoptosis and neurite extension by FKBP38 is required for neural tube formation in the mouse

FKBP38 (also known as FKBP8) is a transmembrane chaperone protein that inhibits apoptosis by recruiting the anti-apoptotic proteins Bcl-2 and Bcl-x(L) to mitochondria. We have now generated mice harboring a loss-of-function mutation in Fkbp38. The Fkbp38(-/-) mice die soon after birth manifesting defects in neural tube closure in the thoraco-lumbar-sacral region (spina bifida) as well as skeletal defects including scoliosis, rib deformities, club foot and curled tail. The neuroepithelium is disorganized and that formation of dorsal root ganglia is defective in Fkbp38(-/-) embryos, likely as a result of an increased frequency of apoptosis and aberrant migration of neuronal cells. Furthermore, the extension of nerve fibers in the spinal cord is abnormal in the mutant embryos. To explore the mechanisms underlying these characteristics, we screened for proteins that interact with FKBP38 in the yeast two-hybrid system and thereby identified protrudin, a protein that promotes process formation by regulating membrane trafficking. Protrudin was found to be hyperphosphorylated in the brain of Fkbp38(-/-) mice, suggesting that FKBP38 regulates protrudin-dependent membrane recycling and neurite outgrowth. Together, our findings suggest that FKBP38 is required for neuroectodermal organization during neural tube formation as a result of its anti-apoptotic activity and regulation of neurite extension.

Proper Gcn5 histone acetyltransferase expression is required for normal anteroposterior patterning of the mouse skeleton

Histone acetylation plays important roles in gene regulation. However, the functions of individual histone acetyltransferases (HATs) in specific developmental transcription programs are not well defined. To define the functions of Gcn5, a prototypical HAT, during mouse development, we have created a series of mutant Gcn5 alleles. Our previous work revealed that deletion of Gcn5 leads to embryonic death soon after gastrulation. Embryos homozygous for point mutations in the catalytic center of Gcn5 survive longer, but die soon after E16.0 and exhibit defects in cranial neural tube closure. Embryos bearing a hypomorphic Gcn5(flox(neo)) allele also exhibit neural closure defects and die at or soon after birth. We report here that Gcn5(flox(neo)/flox(neo)) and Gcn5(flox(neo)/Delta) embryos exhibit anterior homeotic transformations in lower thoracic and lumbar vertebrae. These defects are accompanied by a shift in the anterior expression boundary of Hoxc8 and Hoxc9. These data provide the first evidence that Gcn5 contributes to Hox gene regulation and is required for normal anteroposterior patterning of the mouse skeleton.

Proper expression of the Gcn5 histone acetyltransferase is required for neural tube closure in mouse embryos

Histone acetyltransferases (HATs) are important to gene activation, altering chromatin structures to facilitate association of transcription proteins with gene promoters. The functions of individual HATs in mammalian developmental are not well defined. Our previous studies demonstrated that Gcn5, a prototypical HAT, is required for mesodermal maintenance in early embryos. Homozygous Gcn5 null embryos die soon after gastrulation, preventing determination of Gcn5 functions later during development. We report here the creation of a Gcn5(flox(neo)) allele, which is only partially functional and gives rise to a hypomorphic phenotype. Mice homozygous for this allele had an increased risk of cranial neural tube closure defects (NTDs) and exencephaly. These defects were found at an even greater penetrance in Gcn5(flox(neo)/Delta) embryos. These results indicate that normal levels of Gcn5 expression are critical for neural tube closure in mice and predict that mutations in this HAT may be associated with increased risk of NTDs in humans.

Connecting Wnt to the Cytoskeleton

The planar cell polarity pathway is important for the proper orientation of cells in various tissues. Wnt is an extracellular signal that triggers this process by stimulating rearrangement of the actin cytoskeleton through the actions of the guanine triphosphatases (GTPases) Rho and Rac. However, the guanine nucleotide exchange factors (GEFs) that connect Wnt signaling to these GTPases are unknown. While screening Xenopus embryos for genes expressed in the notochord--a tissue that undergoes convergent extension, which is a process that mediates the anterior-posterior extension of the body axis during development--Tanegashima et al . identified a gene homologous to human WGEF (encoding weak-similarity GEF). XWGEF was expressed at the early gastula stage, became restricted to the notochord at the end of gastrulation, and then in the tail-bud stages expression was detected in the notochord and head region. When a tagged version of XWGEF was overexpressed, the protein was found colocalized with actin and appeared to be associated with the cell membrane. When expressed in human 293 cells, XWGEF coimmunoprecipitated and increased the abundance of GTP-bound RhoA but did not interact with and had no effect on Rac or Cdc42. When mRNA for hWGEF or XWGEF was injected into Xenopus embryos, RhoA activation was observed and the embryos exhibited phenotypes similar to those observed in which a constitutively active RhoA mRNA was injected. Injection of mRNA encoding a dominant-negative RhoA or a dominant-negative Rok (a downstream effector of RhoA), but not mRNA encoding a dominant-negative Rac1, rescued the body axis phenotype produced by overexpression of XWGEF. Using morpholino oligonucleotides to reduce XWGEF, the authors found that the embryos exhibited neural tube closure defects and a shortened axis, consistent with a defect in convergent extension. Epistasis experiments placed XWGEF downstream of Wnt and upstream of Daam-1 (a formin-homology protein). Tagged proteins expressed in mammalian cultured cells showed that WGEF interacted with both Dishevelled (Dsh, a component downstream of Wnt) and Daam-1. The authors propose a model whereby activation of the Frizzled receptor by Wnt leads to the formation of a complex of Dsh, Daam-1, and WGEF at the membrane, which allows WGEF to stimulate RhoA and regulate the actin cytoskeleton. K. Tanegashima, H. Zhao, I. B. Dawid, WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in Xenopus gastrulation. EMBO J. 27 , 606-617 (2008). [PubMed]