Ultrastructural observations on closure of the neural tube in the mouse

Summary The fusion of the neural walls in the cephalic part of mouse embryos varying in age from 9 to 20 somites was examined with the electron microscope. In the rhombencephalic region the rim of the neural wall was formed from outside inward by ectodermal surface cells, a row of flattened cells without surface projections and neuroepithelial cells. At the junction of the surface ectoderm and the flat cells were seen large projections containing a cytoplasmic matrix without organelles and previously referred to as ruffles. The initial contact between the walls was made by the large cytoplasmic arms and numerous finger-like projections interdigitating with similar projections from the opposite wall. The projections originated from the surface ectoderm and possibly neural crest cells. During further fusion the surface ectoderm cells formed dense membrane specializations, thus establishing a firm contact.The initial contact in the mesencephalon was formed by extensions from the surface ectoderm and was followed by the formation of specialized membrane junctions, as seen between the surface ectoderm in the rhombencephalon. The neuroepithelial cells facing the gap between the neural walls with their apical ends made contact with the cells from the opposing wall by numerous finger-like projections but membrane specializations failed to develop.The closing mechanism in the prosencephalon and anterior neuropore regions differed from the previous areas in that the initial contact was established by the neuroepithelial cells. Only after this contact had been formed did the surface ectoderm cells close the gap. In contrast with the other areas many phagocytosed particles were seen in the prosencephalon and in the region of the anterior neuropore. Many particles from degenerated cells were found inside healthy surrounding cells. Some of these particles contained nuclear material and cytoplasmic organelles.     

Bmp2 is required for cephalic neural tube closure in the mouse

BMPs have been shown to play a role in neural tube development particularly as dorsalizing factors. To explore the possibility that BMP2 could play a role in the developing neural tube (NT) beyond the lethality of Bmp2 null embryos, we created Bmp2 chimeras from Bmp2 null ES cells and WT blastocysts. Analysis of Bmp2 chimeras reveals NT defects at day 9.5 (E9.5). We found that exclusion of Bmp2 null ES cells from the dorsal NT did not always prevent defects. For further comparison, we used a Bmp2 mutant line in a mixed background. Phenotypes observed were similar to chimeras including open NT defects, postneurulation defects, and abnormal neural ectoderm in heterozygous and homozygous null embryos demonstrating a pattern of dose‐dependent signaling. Our data exposes BMP2 as a unique player in the developing NT for dorsal patterning and identity, and normal cephalic neural tube closure in a dose‐dependent manner. Developmental Dynamics 238:110–122, 2009. Published 2008 Wiley‐Liss, Inc.     

A comparison between trisomy 12 and vitamin A induced exencephaly and associated malformations in the mouse embryo

Summary Morphological and morphometric comparison of exencephaly in 13–15 day old mouse embryos caused either by chromosomal trisomy (Ts 12) or the teratogenic action of Vitamin A disclosed significant similarities as well as distinct differences: similarities included an open cranial vault and laterally everted brain, shortening of the sagittal and transversal axis of the skull base, caudal shift of the optic axis, deformation and dislocation of the hypophysis and inner ear due to a change of growth vectors of the skull base and its surrounding tissue. The differences were that in Ts 12 the caudal shift of the eyes causes enophthalmia, while after Vitamin A application additional malformations of the facial bones and defective formation of the orbits result in exophthalmia; the inner ear shows developmental retardation and hypoplasia only in Ts 12, but severe impairment of the development of all parts of the ear with complete lack of the outer and middle ear characterizes Vitamin A damage. Similarly, marked inhibition of hypophyseal development, the residues of Rathke's pouch, and cleft of the basal sphenoid occurs after Vitamin A, but is absent in Ts 12.The differences in the exencephalic malformation complex suggest that the area affected in early organogenesis which determins the later malformation, is limited to the paraxial mesoderm in Ts 12, whereas Vitamin A treatment produces additional head mesenchyme abnormalities, in particular of neural crest derivatives. Although Vitamin A-damaged embryos show more extensive malformations, Ts 12 embryos have a shorter lifespan. This is a consequence of the fact that all the cells of the embryo are endowed with the abnormal chromosomal condition.Supported by a Grant (Gr 71/43) of the Deutsche Forschungsgemeinschaft to A.G. Part of this work was done in fulfilment of Contr. NICHD-NO1-HD-8-2858 (A.G.)     

Mammalian embryos show metabolic plasticity toward the surrounding environment during neural tube closure

Developing embryos rewire energy metabolism for developmental processes. However, little is known about how metabolic rewiring is coupled with development in a spatiotemporal manner. Here, we show that mammalian embryos display plasticity of glucose metabolism in response to the extracellular environment at the neural tube closure (NTC) stage, when the intrauterine environment changes upon placentation. To study how embryos modulate their metabolic state upon environmental change, we analyzed the steady‐state level of ATP upon exposure to extrauterine environments using both an enzymatic assay and a genetically encoded ATP sensor. Upon environmental changes, NTC‐stage embryos exhibited increased ATP content, whereas embryos before and after NTC did not. The increased ATP in the NTC‐stage embryos seemed to depend on glycolysis. Intriguingly, an increase in mitochondrial membrane potential (ΔΨm) was also observed in the neural ectoderm (NE) and the neural plate border of the non‐neural ectoderm (NNE) region. This implies that glycolysis can be coupled with the TCA cycle in the NE and the neural plate border depending on environmental context. Disrupting ΔΨm inhibited folding of the cranial neural plate. Thus, we propose that embryos tune metabolic plasticity to enable coupling of glucose metabolism with the extracellular environment at the NTC stage.     

母体肥胖对胚胎神经管发育及线粒体结构和功能的影响

目的:研究母体肥胖对胚胎神经管发育及线粒体结构和功能的影响.方法:将16只C57BL/6J雌性小鼠随机分为正常饮食(normal diet,ND)组和高脂饮食(high-fat diet,HFD)组,连续喂食12周后,使雌鼠受孕,于孕14.5 d时进行后续实验.检测小鼠体重、血脂,行肝脏HE和油红O染色以判断肥胖模型是...     

Single‐site neural tube closure in human embryos revisited

Since the multi‐site closure theory was first proposed in 1991 as explanation for the preferential localizations of neural tube defects, the closure of the neural tube has been debated. Although the multi‐site closure theory is much cited in clinical literature, single‐site closure is most apparent in literature concerning embryology. Inspired by Victor Hamburgers (1900–2001) statement that “our real teacher has been and still is the embryo, who is, incidentally, the only teacher who is always right”, we decided to critically review both theories of neural tube closure. To verify the theories of closure, we studied serial histological sections of 10 mouse embryos between 8.5 and 9.5 days of gestation and 18 human embryos of the Carnegie collection between Carnegie stage 9 (19–21 days) and 13 (28–32 days). Neural tube closure was histologically defined by the neuroepithelial remodeling of the two adjoining neural fold tips in the midline. We did not observe multiple fusion sites in neither mouse nor human embryos. A meta‐analysis of case reports on neural tube defects showed that defects can occur at any level of the neural axis. Our data indicate that the human neural tube fuses at a single site and, therefore, we propose to reinstate the single‐site closure theory for neural tube closure. We showed that neural tube defects are not restricted to a specific location, thereby refuting the reasoning underlying the multi‐site closure theory. Clin. Anat. 30:988–999, 2017. © 2017 Wiley Periodicals, Inc.     

The first appearance of the neural tube and optic primordium in the human embryo at stage 10

Summary Thirteen embryos of stage 10 (22 days) were studied in detail and graphic reconstructions of most of them were prepared. The characteristic feature of this stage is 4–12 pairs of somites. Constantly present are the prechordal and notochordal plates (the notochord sensu stricto is not yet apparent), the neurenteric canal or at least its site, the thyroid primordium, probably the mesencephalic and rhombencephalic neural crest and the adenohypophysial primordium. During this stage, the following features appear: terminal notch, optic sulcus, initial formation of neural tube, oropharyngeal membrane, pulmonary primordium, cardiac loop, aortic arches 1–3, intersegmental arteries, and laryngotracheal groove. The primitive streak is still an important feature.Graphic reconstructions have permitted the detection of the telencephalic portion of the forebrain, for the first time at such an early stage. It is proposed that the remainder of the forebrain comprises two subdivisions: D1, which becomes largely the optic primordium during stage 10, and D2, which is the future thalamic region. The optic sulcus is found in D1 but does not extent into D2, as has been claimed in the literature. An indication of invagionation of the otic disc appears towards the end of the stage. As compared with the previous stage, the prosencephalon has increased in length, the mesencephalon has remained the same, the rhombencephalon has decreased, and the spinal part of the neural plate has increased fivefold in length. The site of the initial closure of the neural groove is rhombencephalic, upper cervical, or both. The neural plate extends caudally beyond the site of the neurenteric canal. Cytoplasmic inclusions believed to indicate locations of great activity were always detected in the forebrain (especially in the optic primordium), and also in the rhombencephalon, spmal part, and mesencephalon.Supported by research grant No. HD-16702, Institute of Child Health and Human Development, National Institutes of Health (USA)     

孕妇血清叶酸、维生素B12和同型半胱氨酸水平与胎儿神经管畸形相关性研究

目的比较胎儿神经管畸形（neural tube defects,NTDs）孕妇和正常孕妇血清叶酸（Folate,Fol）、维生素B12（Vitamin B12,B12）和同型半胱氨酸（homocysteine,Hcy）水平,以了解三种物质的代谢水平与胎儿NTDs的关系。方法根据有无胎儿NTDs将研究对象分为病例组（n=47）和对照组（n=43）,两组共90例。病例组指经B超诊断为孕NTDs胎儿并通过终止妊娠确诊活产或死产NTDs胎儿的孕妇。对照组指经B超诊断胎儿发育正常,并经随访确认所分娩婴儿正常的孕妇中随机抽取43例。检测两组孕妇血清标本Fol、B12和Hcy水平,并比较两组孕妇血清Fol、B12和Hcy水平差异。结果病例组和对照组血清Hcy水平分别为9.31±4.19（8.08~10.54）μmol/L和7.75±1.95（7.15~8.35）μmol/L,两组经独立样本t检验比较,差异有统计学意义（t=2.189,P=0.031）;两组血清Fol和B12水平比较,差异均无统计学意义（P〉0.05）。单因素Logistic回归分析从6个变量中遴选出1个危险因素,Hcy OR=2.021（95%CI：1.167~3.500）,是子代发生NTDs的危险因素;多因素Logistic回归分析结果显示,随着孕妇血清Hcy水平的升高,其发生胎儿NTDs发病风险增加。结论中孕期妇女血清高水平Hcy是妊娠NTDs的独立危险因素,随着孕妇血清Hcy水平的升高,其发生胎儿NTDs发病风险增加。     

Evidence for multi-site closure of the neural tube in humans

Four separate initiation sites for neural tube (NT) fusion have been demonstrated recently in mice and other experimental animals. We evaluated the question of whether the mult-site model vs. the traditional single-site model of NT closure provided the best explanation for neural tube defects (NTDs) in humans. Evidence for segmental vs. continuous NT closure was obtained by review of our recent clinical cases of NTDs and previous medical literature. With the multi-site NT closure model, we find that the majority of NTDs can be explained by failure of fusion of one of the closures or their contiguous neuropores. We hypothesize that: Anencephaly results from failure of closure 2 for meroacranium and closures 2 and 4 for holoacranium. Spina bifida cystica results from failure of rostral and/or caudal closure 1 fusion. Craniorachischisis results from failure of closures 2,4, and 1. Closure 3 non-fusion is rare, presenting as a midfacial cleft extending from the upper lip through the frontal area (“facioschisis”). Frontal and parietal cephaloceles occur at the sites of the junctions of the cranial closures 3-2 and 2–4 (the prosencephalic and mesencephalic neuropores). Occipital cephaloceles result from incomplete membrane fusion of closure 4. In humans, the most caudal NT may have a 5th closure site involving L2 to S2. Closure below S2 is by secondary neurulation. Evidence for multi-site NT closure is apparent in clinical cases of NTDs, as well as in previous epidemiological studies, empiric recurrence risk studies, and pathological studies. Genetic variations of NT closures sites occur in mice and are evident in humans, e.g., familial NTDs with Sikh heritage (closure 4 and rostral 1), Meckel-Gruber syndrome (closure 4), and Walker-Warburg syndrome (2–4 neuropore, closure 4). Environmental and teratogenic exposures frequently affect specific closure sites, e.g., folate deficiency (closures 2, 4, and caudal 1) and valproic acid (closure 5 and canalization). Classification of NTDs by closure site is recommended for all studies of NTDs in humans. We conclude that (1) multi-site NT closure provides the best explanation for NTDs in humans; (2) closure sites are most likely controlled by separate genes expressed during embryogenesis, and variations in rate and location of closures would make embryos more susceptible to environmental and other factors; (3) homologies between the mouse and human genomes may allow linkage studies to be done for families with recurrence of NTDs; and (4) recurrence risks and associated anomalies vary between closure sites, so that more accurate genetic counselling can be given based on the location of the NTD(s) in the proband(s). © 1993 Wiley-Liss, Inc.     

神经管畸形动物模型的研究进展

神经管畸形(neural tube defects,NTDs)是一类常见的发病率高且后果严重的出生缺陷,为影响我国出生人口素质的重要问题.目前,其大部分研究内容依靠动物模型来完成.神经管畸形动物模型的建立对研究其发病机制及其防治起着重要作用.本文从物理和化学两方面因素对神经管畸形动物模型诱导方法及其机制作一综述.     

Amniotic fluid homocysteine levels, 5,10‐methylenetetrahydrafolate reductase genotypes,and neural tube closure sites

A specific gene mutation leading to altered homocysteine metabolism has been identified in parents and fetuses with neural tube defects (NTDs). In addition, current animal and human data indicate that spine closure occurs simultaneously in five separate sites that then fuse. We sought to determine whether either this mutation or abnormal amniotic fluid homocysteine levels are associated with all five neural tube closure sites. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80) and from normal controls matched for race, month and year of amniocentesis, and maternal age. Cases were categorized according to defect site by using all available medical records. The presence or absence of the 677C→T mutation of 5,10‐methylenetetrahydrafolate reductase (MTHFR) gene was determined, and homocysteine levels were measured; case and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C→T MTHFR mutation (44% vs. 17%, P ≤ 0.001). Likewise, cases were significantly more likely than controls to have amniotic fluid homocysteine levels >90th centile (>1.85 μmol/L), 27% vs. 10%, P = 0.02. Most (83%) of control cases had both normal MTHFR alleles and normal amniotic fluid homocysteine levels (normal/normal), whereas only 56% of NTD case were normal/normal (P = 0.001). When evaluated by defect site, only defects involving the cervical‐lumbar spine, lumbosacral spine, and occipital encephalocele were significantly less likely to be normal/normal than controls (P = 0.007, 0.0003, and 0.007, respectively), suggesting a strong association with the 677C→T allele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum included many cases that had both normal MTHFR alleles and normal homocysteine and were not significantly different from controls. The 677C→T MTHFR mutation and elevated homocysteine levels appear to be disproportionately associated with defects spanning the cervical‐lumbar spine, lumbosacral spine, and occipital encephalocele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum may not be related to altered homocysteine metabolism. Am. J. Med. Genet. 90:6–11, 2000. © 2000 Wiley‐Liss, Inc.     

神经管畸形的预防

NTDs为多因素疾病,发病率高,危害严重.现有的预防措施主要有围孕期服用叶酸制剂及产前监测.孕前筛查利用基因诊断筛查出生育NTDs的亲代高危人群,为孕前重点干预及孕期重点监测提供依据.若3种方法结合可使NTDs的预防措施更具针对性,从而最大限度地有效降低NTDs的发生率.     

Malformations of the axial skeleton in cranioschisis aperta and exencephaly in rat fetuses induced after neural tube closure

Summary Single doses of cyclophosphamide were administered (IP) to groups of Wistar rats, on different days of gestation after neural tube closure (days 12–15) and fetuses were collected on day 20. A very large number of the fetuses treated during days 12–14 exhibited cranioschisis and exencephaly. Ethanol fixed, alizarin red stained specimens were observed for axial skeletal abnormalities. The exencephalic ones lacked ossified skulls. The basicranial bones were either under ossified or had undergone extensive fusion resulting in reduction in cranial volume. The basicraniovertebral angle was obtuse. The vertebral bodies and arches showed varying degrees of hypoplasia, fusion and/or agenesis. The development of ribs and sternebrae was also extensively affected. Treatment on day 15 did not induce exencephaly but the axial skeleton was hypoplastic. Wavy ribs were a remarkable feature of these fetuses. All exencephalic fetuses had subcutaneous haemorrhages; many of them were obviously oedematous. These data indicate that the susceptible period for induction of these anomalies does not stop at neuropore closure.     

Effect of the notochord on the differentiation of a floor plate area in the neural tube of the chick embryo

Summary The role of a notochord fragment on the origin of an additional floor plate area in the neural tube is investigated by quantitative morphological methods. In 1.5 to 2 day chick embryos a notochordal fragment was implanted in close apposition to the lateral wall of the neural groove in the region between prospective wing and leg bud. At 4 days, adjacent to the implant a distinct area of the neural wall was present, which resembled the natural floor plate with respect to its thickness, the abluminal location of elongated nuclei and the absence of neuroblasts. The mitotic density of this area was reduced. This additional floor plate was distinct when the experiment was performed at 1.5 days but was hardly recognizable when it was carried out at 2 days.From these results it is concluded that a) the notochord induces floor plate like structures and diminishes proliferation, and b) that the period of floor plate induction by the notochord is very restricted.     

被动吸烟诱导金黄地鼠神经管形成过程中细胞过度死亡

目的研究被动吸烟对胎鼠神经系统发育的影响,探讨其致神经管缺陷的可能机制.方法将怀孕的金黄地鼠用烟熏的方法建立被动吸烟的致畸模型,在体视显微镜下观察鼠胚的发育情况;采用甲苯胺蓝染色技术,观察被动吸烟对胎鼠神经上皮细胞死亡和增殖的影响.结果被动吸烟对胚胎有显著的致畸作用(χ2 =51.28,P<0.01).吸烟组固缩细胞数明显高于对照组(F=21.95,P<0.01),随着胎龄的增加固缩细胞数也有所增加(F=21.49,P<0.01);吸烟组的有丝分裂相计数高于对照组(F=11.91,P<0.01),随着胎龄的增加有丝分裂相数也有所增加(F=70.65,P<0.01).结论被动吸烟可诱导神经上皮及其周围间充质细胞变性、死亡,导致了神经管的形成和分化异常,使神经管延迟闭合或不闭合.香烟烟雾能提高有丝分裂率,可以部分代偿由其引起的神经上皮细胞的减少,但不能弥补由于细胞过度死亡而导致神经管缺陷.     

Sex,neural tube defects,and multisite closure of the human neural tube

While neural tube defects (NTD) overall have a female sex bias, this does not apply to all sites along the neuraxis. The findings regarding sex and NTD in a series of midtrimester fetuses are reviewed, and then analysed in terms of the recent hypothesis that during embryogenesis of the human neural tube there are multiple closure sites, rather than a single zipping up process. Females more often than males tend to have craniorachischisis, spina bifida involving the thorax, the holoacrania form of anencephaly, anencephaly and cervical spina bifida and encephalocoeles, while males more often than females have spina bifida affecting the lower spine. Meroacrania occurs equally in both sexes. Other sources indicate that there is a male bias in frontoethmoidal encephalocoeles. Since sex seems to be a factor that is differentially associated with lack of closure of specific areas of the neural tube, it would seem to support the notion that there are multiple closure sites in the human neural tube. However, no association was found between a particular sex and either the type of NTD which have an isolated abnormality or those NTD associated with developmental abnormalities of other body systems. © 1995 Wiley-Liss, Inc.     

Autotaxin is required for the cranial neural tube closure and establishment of the midbrain–hindbrain boundary during mouse development

Autotaxin (ATX) is a lysophospholipid‐generating exoenzyme expressed in embryonic and adult neural tissues. We previously showed that ATX is expressed in the neural organizing centers, anterior head process, and midbrain‐hindbrain boundary (MHB). To elucidate the role of ATX during neural development, here we examined the neural phenotypes of ATX‐deficient mice. Expression analysis of neural marker genes revealed that lateral expansion of the rostral forebrain is reduced and establishment of the MHB is compromised as early as the late headfold stage in ATX mutant embryos. Moreover, ATX mutant embryos fail to complete cranial neural tube closure. These results indicate that ATX is essential for cranial neurulation and MHB establishment. Developmental Dynamics 240:413–421, 2011. © 2011 Wiley‐Liss, Inc.     

Relationship between altered axial curvature and neural tube closure in normal and mutant (curly tail) mouse embryos

Neural tube defects, including spina bifida, develop in the curly tail mutant mouse as a result of delayed closure of the posterior neuropore at 10.5 days of gestation. Affected embryos are characterized by increased ventral curvature of the caudal region. To determine whether closure of the neuropore could be affected by this angle of curvature, we experimentally enhanced the curvature of non-mutant embryos. The amnion was opened in 9.5 day embryos; after 20 h of culture, a proportion of the embryos exhibited a tightly wrapped amnion with enhanced curvature of the caudal region compared with the control embryos in which the opened amnion remained inflated. Enhanced curvature correlated with a higher frequency of embryos with an open posterior neuropore, irrespective of developmental stage within the range, 27–32 somites. Thus, within this somite range, caudal curvature is a more accurate determinant for normal spinal neurulation than the exact somite stage. Enhanced ventral curvature of the curly tail embryo correlates with an abnormal growth difference between the neuroepithelium and ventral structures (the notochord and hindgut). We experimentally corrected this imbalance by culturing under conditions of mild hyperthermia and subsequently determined whether the angle of curvature would also be corrected. The mean angle of curvature and length of the posterior neuropore were both reduced in embryos cultured at 40.5°C by comparison with control embryos cultured at 38°C. We conclude that the sequence of morphogenetic events leading to spinal neural tube defects in curly tail embryos involves an imbalance of growth rates, which leads to enhanced ventral curvature that, in turn, leads to delayed closure of the posterior neuropore.