Valproic acid disrupts the biomechanics of late spinal neural tube closure in mouse embryos

Failure of neural tube closure in the early embryo causes neural tube defects including spina bifida. Spina bifida lesions predominate in the distal spine, particularly after exposure to the anticonvulsant valproic acid (VPA). How VPA specifically disturbs late stages of neural tube closure is unclear, as neurulation is usually viewed as a uniform 'zippering' process along the spine. We recently identified a novel closure site ("Closure 5") which forms at the caudal extremity of the mouse posterior neuropore (PNP) when completion of closure is imminent. Here we investigated whether distal spina bifida in VPA-exposed embryos involves disruption of Closure 5. Exposure of E8.5 mouse embryos to VPA in whole embryo culture had marked embryotoxic effects, whereas toxic effects were less pronounced in more developmentally advanced (E9) embryos. Only 33% of embryos exposed to VPA from E9 to E10.5 achieved PNP closure (control=90%). Short-term (8h) VPA treatment diminished supra-cellular F-actin cables which normally run along the lateral neural folds, and prevented caudal PNP narrowing normally characteristic of Closure 5 formation. Laser ablation of Closure 5 caused rapid neuropore widening. Equivalent ablations of the caudal PNP in VPA treated embryos resulted in significantly less widening, suggesting VPA prevents formation of Closure 5 as a biomechanically active structure. Thus, VPA exposure prevents morphological and biomechanical conversion of the caudal extreme of the PNP during late spinal closure. Closure 5 facilitates neural fold apposition when completion of closure is imminent, such that its disruption in VPA-exposed embryos may lead to distal spina bifida

Neural tube closure: cellular, molecular and biomechanical mechanisms.

Neural tube closure has been studied for many decades, across a range of vertebrates, as a paradigm of embryonic morphogenesis. Neurulation is of particular interest in view of the severe congenital malformations - 'neural tube defects' - that result when closure fails. The process of neural tube closure is complex and involves cellular events such as convergent extension, apical constriction and interkinetic nuclear migration, as well as precise molecular control via the non-canonical Wnt/planar cell polarity pathway, Shh/BMP signalling, and the transcription factors Grhl2/3, Pax3, Cdx2 and Zic2. More recently, biomechanical inputs into neural tube morphogenesis have also been identified. Here, we review these cellular, molecular and biomechanical mechanisms involved in neural tube closure, based on studies of various vertebrate species, focusing on the most recent advances in the field

Top Quarks as a Window to String Resonances

We study the discovery potential of string resonances decaying to $t\bar{t}$ final state at the LHC. We point out that top quark pair production is a promising and an advantageous channel for studying such resonances, due to their low Standard Model background and unique kinematics. We study the invariant mass distribution and angular dependence of the top pair production cross section via exchanges of string resonances. The mass ratios of these resonances and the unusual angular distribution may help identify their fundamental properties and distinguish them from other new physics. We find that string resonances for a string scale below 4 TeV can be detected via the $t\bar{t}$ channel, either from reconstructing the $t\bar{t}$ semi-leptonic decay or recent techniques in identifying highly boosted tops.Comment: 22 pages, 6 figure

Prenatal hypoxia induces increased cardiac contractility on a background of decreased capillary density.

Background: Chronic hypoxia in utero (CHU) is one of the most common insults to fetal development and may be associated with poor cardiac recovery from ischaemia-reperfusion injury,yet the effects on normal cardiac mechanical performance are poorly understood. Methods: Pregnant female wistar rats were exposed to hypoxia (12% oxygen, balance nitrogen)for days 10–20 of pregnancy. Pups were born into normal room air and weaned normally. At 10 weeks of age, hearts were excised under anaesthesia and underwent retrograde 'Langendorff' perfusion. Mechanical performance was measured at constant filling pressure (100 cm H2O) with intraventricular balloon. Left ventricular free wall was dissected away and capillary density estimated following alkaline phosphatase staining. Expression of SERCA2a and Nitric Oxide Synthases (NOS) proteins were estimated by immunoblotting. Results: CHU significantly increased body mass (P < 0.001) compared with age-matched control rats but was without effect on relative cardiac mass. For incremental increases in left ventricular balloon volume, diastolic pressure was preserved. However, systolic pressure was significantly greater following CHU for balloon volume = 50 μl (P < 0.01) and up to 200 μl (P < 0.05). For higher balloon volumes systolic pressure was not significantly different from control. Developed pressures were correspondingly increased relative to controls for balloon volumes up to 250 μl (P < 0.05).Left ventricular free wall capillary density was significantly decreased in both epicardium (18%; P <0.05) and endocardium (11%; P < 0.05) despite preserved coronary flow. Western blot analysis revealed no change to the expression of SERCA2a or nNOS but immuno-detectable eNOS protein was significantly decreased (P < 0.001) in cardiac tissue following chronic hypoxia in utero. Conclusion: These data offer potential mechanisms for poor recovery following ischaemia, including decreased coronary flow reserve and impaired angiogenesis with subsequent detrimental effects of post-natal cardiac performance

Stationary Black Holes: Uniqueness and Beyond

The spectrum of known black-hole solutions to the stationary Einstein equations has been steadily increasing, sometimes in unexpected ways. In particular, it has turned out that not all black-hole-equilibrium configurations are characterized by their mass, angular momentum and global charges. Moreover, the high degree of symmetry displayed by vacuum and electro-vacuum black-hole spacetimes ceases to exist in self-gravitating non-linear field theories. This text aims to review some developments in the subject and to discuss them in light of the uniqueness theorem for the Einstein-Maxwell system.Comment: Major update of the original version by Markus Heusler from 1998. Piotr T. Chru\'sciel and Jo\~ao Lopes Costa succeeded to this review's authorship. Significantly restructured and updated all sections; changes are too numerous to be usefully described here. The number of references increased from 186 to 32

Novel mouse model of encephalocele: post-neurulation origin and relationship to open neural tube defects

Encephalocele is a clinically important birth defect that can lead to severe disability in childhood and beyond. The embryonic and early fetal pathogenesis of encephalocele is poorly understood and, while usually classified as a 'neural tube defect', there is conflicting evidence on whether encephalocele results from defective neural tube closure, or is a post-neurulation defect. It is also unclear whether encephalocele can result from the same causative factors as anencephaly and open spina bifida, or whether it is aetiologically distinct. This lack of information results largely from the scarce availability of animal models of encephalocele, particularly ones that resemble the commonest, non-syndromic human defects. Here, we report a novel mouse model of occipito-parietal encephalocele, in which the small GTPase Rac1 is conditionally ablated in the (non-neural) surface ectoderm. Most mutant fetuses have open spina bifida, and some also exhibit exencephaly/anencephaly. However, a proportion of mutant fetuses exhibit brain herniation, affecting the occipito-parietal region and closely resembling encephalocele. The encephalocele phenotype does not result from defective neural tube closure, but rather from a later disruption of the surface ectoderm covering the already closed neural tube, allowing the brain to herniate. The neuroepithelium itself shows no down-regulation of Rac1 and appears morphologically normal until late gestation. A large skull defect overlies the region of brain herniation. Our work provides a new genetic model of occipito-parietal encephalocele, particularly resembling non-syndromic human cases. While encephalocele has a different, later-arising pathogenesis than open neural tube defects, both can share the same genetic causation

Glycine Cleavage System H Protein Is Essential for Embryonic Viability, Implying Additional Function Beyond the Glycine Cleavage System

Glycine cleavage system H protein (GCSH) is a component of the glycine cleavage system (GCS), a conserved protein complex that acts to decarboxylate glycine. Mutation of AMT or GLDC, encoding the GCS components aminomethyltransferase and glycine decarboxylase, can cause malformations of the developing CNS (neural tube defects (NTDs) and ventriculomegaly) as well as a post-natal life-limiting neurometabolic disorder, Non-Ketotic Hyperglycinemia. In contrast, it is unclear whether mutation of GCSH contributes to these conditions and we therefore investigated GCSH loss of function in mice. Mice that were heterozygous for a Gcsh null allele were viable and did not exhibit elevated plasma glycine. Moreover, heterozygous mutation of Gcsh did not increase the frequency of NTDs in Gldc mutant embryos. Homozygous Gcsh null mice were not recovered at post-natal stages. Analysis of litters at E8.5-10.5, revealed the presence of homozygous null embryos which were much smaller than littermates and had failed to develop beyond early post-implantation stages with no visible somites or head-folds. Hence, unlike null mutations of Gldc or Amt, which are compatible with embryonic survival despite the presence of NTDs, loss of Gcsh causes embryonic death prior to mid-gestation. Maternal supplementation with formate did not restore embryonic development beyond E7.5, suggesting that the primary cause of lethality was not loss of glycine cleavage activity or suppression of folate one-carbon metabolism. These findings suggest that GCSH has additional roles beyond function in the glycine cleavage system. We hypothesize that GCSH potentially acts in lipoylation of 2-oxoacid dehydrogenase proteins, as reported in bacteria

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion in mice

Post-zygotic mutations that generate tissue mosaicism are increasingly associated with severe congenital defects, including those arising from failed neural tube closure. Here we report that neural fold elevation during mouse spinal neurulation is vulnerable to deletion of the VANGL planar cell polarity protein 2 (Vangl2) gene in as few as 16% of neuroepithelial cells. Vangl2-deleted cells are typically dispersed throughout the neuroepithelium, and each non-autonomously prevents apical constriction by an average of five Vangl2-replete neighbours. This inhibition of apical constriction involves diminished myosin-II localisation on neighbour cell borders and shortening of basally-extending microtubule tails, which are known to facilitate apical constriction. Vangl2-deleted neuroepithelial cells themselves continue to apically constrict and preferentially recruit myosin-II to their apical cell cortex rather than to apical cap localisations. Such non-autonomous effects can explain how post-zygotic mutations affecting a minority of cells can cause catastrophic failure of morphogenesis leading to clinically important birth defects

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension

Planar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesised this glycosaminoglycan-PCP interaction may regulate CE but, surprisingly, DiO labeling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube

Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies

The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB