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Objective: Neonatal transports are an essential component of regionalized medical systems. Neonates who are unstable after birth require transport to a higher level of care by neonatal transport teams. Data on adverse events on neonatal transports are limited. The aim of this study was to identify, evaluate, and summarize the findings of all relevant studies on adverse events on neonatal transports., Methods: We identified 38 studies reporting adverse events on neonatal transports from January 1, 2000, to December 31, 2019. The adverse events were distributed into 5 categories: vital sign abnormalities, laboratory value abnormalities, equipment challenges, system challenges, cardiopulmonary resuscitation, and transport-related mortality., Results: Most of the evidence surrounds vital sign abnormalities during transport (n = 28 studies), with hypothermia as the most frequently reported abnormal vital sign. Fourteen studies addressed laboratory abnormalities, 12 reported on events related to equipment issues, and 4 reported on system issues that lead to adverse events on transport. Of the 38 included studies, 12 included mortality related to transport as an outcome, and 4 reported on cardiopulmonary resuscitation during transport. There were significant variations in samples, definitions of adverse events, and research quality., Conclusion: Adverse events during neonatal transport have been illuminated in various ways, with vital sign abnormalities most commonly explored in the literature. However, considerable variation in studies limits a clear understanding of the relative frequencies of each type of adverse event. The transport safety field would benefit from more efforts to standardize adverse event definitions, collect safety data prospectively, and pool data across larger care systems., (Copyright © 2023 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to characterize vital sign abnormalities, trajectories, and related risk factors during neonatal transport., Methods: We performed a retrospective analysis of neonates transported within a US regional care network in 2020 to 2021. Demographic and clinical data were collected from electronic records. Group-based trajectory modeling was applied to identify groups of neonates who followed distinct vital sign trajectories during transport. Patients with conditions likely to impact the assessed vital were excluded. Risk factors for trajectories were examined using modified Poisson regression models., Results: Of the 620 neonates in the study, 92% had one abnormal systolic blood pressure (SBP) measure, approximately half had an abnormal heart rate (47%) or temperature (56%), and 28% had an abnormal oxygen saturation measure during transport. Over half (53%) were in a low and decreasing SBP trajectory, and 36% were in a high and increasing heart rate trajectory. Most infants ≤ 28 weeks postmenstrual age had 2 or more concerning vital sign trajectories during transport., Conclusion: Abnormal vital signs were common during neonatal transport, and potentially negative trajectories in heart rate and SBP were more common than temperature or oxygen saturation. Transport teams should be trained and equipped to detect concerning trends and respond appropriately while en route., (Copyright © 2022 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.)

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

Neural tube defects (NTDs) are among the most common and severe congenital malformations and result from incomplete closure of the neural tube during early development. Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) has been suggested to be a risk factor for NTDs and previous studies imply that the mechanism underlying the association between PAH exposure and NTDs may involve oxidative stress and apoptosis. The objectives of this study were to investigate whether there is a direct effect of maternal benzo[α] pyrene (BaP) exposure on the closure of the neural tube in mice, and to examine the underlying mechanisms by combining animal experiments and human subject studies. We found that intraperitoneal injection of BaP from embryonic day 7 at a dose of 250 mg kg -1 induced NTDs (13.3% frequency) in ICR mice. BaP exposure significantly increased expression of genes associated with oxidative stress, Cyp1a1, Sod1 and Sod2, while repressing Gpx1. Elevated apoptosis and higher protein expression of cleaved caspase-3 in the neuroepithelium of treated embryos were observed. Pre-treatment with vitamin E, added to food, significantly protected against BaP-induced NTDs (1.4% frequency) (P < 0.05). Vitamin E also partly normalized oxidative stress related gene expression and excess apoptosis in BaP-treated embryos. Examination of human neural tissues revealed that increased levels of protein carbonyl and apoptosis were related with maternal exposure to PAHs and the risk of NTDs. Collectively, these results suggest that BaP exposure could induce NTDs and that this may involve increased oxidative stress and apoptosis, while vitamin E may have a protective effect., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: We used non-invasive high-frequency ultrasound (HFUS) imaging to investigate embryonic brain development in a mouse model for neural tube defects (NTDs) and non-ketotic hyperglycinemia (NKH)., Method: Using HFUS, we imaged embryos carrying loss of function alleles of Gldc encoding glycine decarboxylase, a component of the glycine cleavage system in mitochondrial folate metabolism, which is known to be associated with cranial NTDs and NKH in humans. We serially examined the same litter during the second half of embryonic development and quantified cerebral structures. Genotype was confirmed using PCR. Histology was used to confirm ultrasound findings., Results: High-frequency ultrasound allowed in utero detection of two major brain abnormalities in Gldc-deficient mouse embryos, cranial NTDs (exencephaly) and ventriculomegaly (corresponding with the previous finding of post-natal hydrocephalus). Serial ultrasound allowed individual embryos to be analysed at successive gestational time points. From embryonic day 16.5 to 18.5, the lateral ventricle volume reduced in wild-type and heterozygous embryos but increased in homozygous Gldc-deficient embryos., Conclusion: Exencephaly and ventriculomegaly were detectable by HFUS in homozygous Gldc-deficient mouse embryos indicating this to be an effective tool to study CNS development. Longitudinal analysis of the same embryo allowed the prenatal onset and progression of ventricle enlargement in Gldc-deficient mice to be determined. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd., (© 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.)

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., (© 2017. Published by The Company of Biologists Ltd.)

Susceptibility to neural tube defects (NTDs), such as anencephaly and spina bifida is influenced by genetic and environmental factors including maternal nutrition. Maternal periconceptional supplementation with folic acid significantly reduces the risk of an NTD-affected pregnancy, but does not prevent all NTDs, and "folic acid non-responsive" NTDs continue to occur. Similarly, among mouse models of NTDs, some are responsive to folic acid but others are not. Among nutritional factors, inositol deficiency causes cranial NTDs in mice while supplemental inositol prevents spinal and cranial NTDs in the curly tail (Grhl3 hypomorph) mouse, rodent models of hyperglycemia or induced diabetes, and in a folate-deficiency induced NTD model. NTDs also occur in mice lacking expression of certain inositol kinases. Inositol-containing phospholipids (phosphoinositides) and soluble inositol phosphates mediate a range of functions, including intracellular signaling, interaction with cytoskeletal proteins, and regulation of membrane identity in trafficking and cell division. Myo-inositol has been trialed in humans for a range of conditions and appears safe for use in human pregnancy. In pilot studies in Italy and the United Kingdom, women took inositol together with folic acid preconceptionally, after one or more previous NTD-affected pregnancies. In nonrandomized cohorts and a randomized double-blind study in the United Kingdom, no recurrent NTDs were observed among 52 pregnancies reported to date. Larger-scale fully powered trials are needed to determine whether supplementation with inositol and folic acid would more effectively prevent NTDs than folic acid alone. Birth Defects Research 109:68-80, 2017. © 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc., (© 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc.)

Microtubules (MTs) are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of MTs, their effects on cell shape and polarity, and their role in large-scale morphogenesis remain poorly understood. Here, these relationships were examined with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate; and cavitation of the teleost neural rod. The latter process has been compared with 'secondary' neurulation that generates the caudal spinal cord in mammals. MTs align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration, which indirectly impacts on cell shape. Whether MTs play other functional roles in mammalian neurulation remains unclear. In the zebrafish, MTs are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric MT apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of MT functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish 'cause' from 'effect'. Future developments will likely rely on novel ways to selectively impair MT function in order to investigate the roles they play., (© 2016 Anatomical Society.)

The EphA4 receptor tyrosine kinase is involved in numerous cell-signalling activities during embryonic development. EphA4 has the ability to bind to both types of ephrin ligands, the ephrinAs and ephrinBs. The C57BL/6J-Epha4rb-2J/GrsrJ strain, denoted Epha4(rb-2J/rb-2J), is a spontaneous mouse mutant that arose at The Jackson Laboratory. These mutants exhibited a synchronous hind limb locomotion defect or "hopping gait" phenotype, which is also characteristic of EphA4 null mice. Genetic complementation experiments suggested that Epha4(rb-2J) corresponds to an allele of EphA4, but details of the genomic defect in this mouse mutant are currently unavailable. We found a single base-pair deletion in exon 9 resulting in a frame shift mutation that subsequently resulted in a premature stop codon. Analysis of the predicted structure of the truncated protein suggests that both the kinase and sterile α motif (SAM) domains are absent. Definitive determination of genotype is needed for experimental studies of mice carrying the Epha4(rb-2J) allele, and we have also developed a method to ease detection of the mutation through RFLP. Eph-ephrin family members are reportedly expressed as numerous isoforms. Hence, delineation of the specific mutation in EphA4 in this strain is important for further functional studies, such as protein-protein interactions, immunostaining and gene compensatory studies, investigating the mechanism underlying the effects of altered function of Eph family of receptor tyrosine kinases on phenotype.

The curly tail mouse provides a model for neural tube defects (spina bifida and exencephaly) that are resistant to prevention by folic acid. The major ct gene, responsible for spina bifida, corresponds to a hypomorphic allele of grainyhead-like 3 (Grhl3) but the frequency of NTDs is strongly influenced by modifiers in the genetic background. Moreover, exencephaly in the curly tail strain is not prevented by reinstatement of Grhl3 expression. In the current study we found that expression of Mthfd1L, encoding a key component of mitochondrial folate one-carbon metabolism (FOCM), is significantly reduced in ct/ct embryos compared to a partially congenic wild-type strain. This expression change is not attributable to regulation by Grhl3 or the genetic background at the Mthfd1L locus. Mitochondrial FOCM provides one-carbon units as formate for FOCM reactions in the cytosol. We found that maternal supplementation with formate prevented NTDs in curly tail embryos and also resulted in increased litter size. Analysis of the folate profile of neurulation-stage embryos showed that formate supplementation resulted in an increased proportion of formyl-THF and THF but a reduction in proportion of 5-methyl THF. In contrast, THF decreased and 5-methyl THF was relatively more abundant in the liver of supplemented dams than in controls. In embryos cultured through the period of spinal neurulation, incorporation of labelled thymidine and adenine into genomic DNA was suppressed by supplemental formate, suggesting that de novo folate-dependent biosynthesis of nucleotides (thymidylate and purines) was enhanced. We hypothesise that reduced Mthfd1L expression may contribute to susceptibility to NTDs in the curly tail strain and that formate acts as a one-carbon donor to prevent NTDs., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development.

Although peri-conceptional folic acid (FA) supplementation can prevent a proportion of neural tube defects (NTD), there is increasing evidence that many NTD are FA non-responsive. The vitamin-like molecule inositol may offer a novel approach to preventing FA-non-responsive NTD. Inositol prevented NTD in a genetic mouse model, and was well tolerated by women in a small study of NTD recurrence. In the present study, we report the Prevention of Neural Tube Defects by Inositol (PONTI) pilot study designed to gain further experience of inositol usage in human pregnancy as a preliminary trial to a future large-scale controlled trial to evaluate efficacy of inositol in NTD prevention. Study subjects were UK women with a previous NTD pregnancy who planned to become pregnant again. Of 117 women who made contact, ninety-nine proved eligible and forty-seven agreed to be randomised (double-blind) to peri-conceptional supplementation with inositol plus FA or placebo plus FA. In total, thirty-three randomised pregnancies produced one NTD recurrence in the placebo plus FA group (n 19) and no recurrences in the inositol plus FA group (n 14). Of fifty-two women who declined randomisation, the peri-conceptional supplementation regimen and outcomes of twenty-two further pregnancies were documented. Two NTD recurred, both in women who took only FA in their next pregnancy. No adverse pregnancy events were associated with inositol supplementation. The findings of the PONTI pilot study encourage a large-scale controlled trial of inositol for NTD prevention, but indicate the need for a careful study design in view of the unwillingness of many high-risk women to be randomised.

Bending of the neural plate at paired dorsolateral hinge points (DLHPs) is required for neural tube closure in the spinal region of the mouse embryo. As a step towards understanding the morphogenetic mechanism of DLHP development, we examined variations in neural plate cellular architecture and proliferation during closure. Neuroepithelial cells within the median hinge point (MHP) contain nuclei that are mainly basally located and undergo relatively slow proliferation, with a 7 h cell cycle length. In contrast, cells in the dorsolateral neuroepithelium, including the DLHP, exhibit nuclei distributed throughout the apico-basal axis and undergo rapid proliferation, with a 4 h cell cycle length. As the neural folds elevate, cell numbers increase to a greater extent in the dorsolateral neural plate that contacts the surface ectoderm, compared with the more ventromedial neural plate where cells contact paraxial mesoderm and notochord. This marked increase in dorsolateral cell number cannot be accounted for solely on the basis of enhanced cell proliferation in this region. We hypothesised that neuroepithelial cells may translocate in a ventral-to-dorsal direction as DLHP formation occurs, and this was confirmed by vital cell labelling in cultured embryos. The translocation of cells into the neural fold, together with its more rapid cell proliferation, leads to an increase in cell density dorsolaterally compared with the more ventromedial neural plate. These findings suggest a model in which DLHP formation may proceed through 'buckling' of the neuroepithelium at a dorso-ventral boundary marked by a change in cell-packing density., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The cytoskeleton is widely considered essential for neurulation, yet the mouse spinal neural tube can close despite genetic and non-genetic disruption of the cytoskeleton. To investigate this apparent contradiction, we applied cytoskeletal inhibitors to mouse embryos in culture. Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure. In contrast, inhibiting Rho kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) or blocking F-actin disassembly prevented closure, with apical F-actin accumulation and adherens junction disturbance in the neuroepithelium. Cofilin-1-null embryos yielded a similar phenotype, supporting the hypothesis that there is a key role for actin turnover. Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect. We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly. In contrast, actomyosin assembly and myosin ATPase activity are not limiting for closure., (© 2015. Published by The Company of Biologists Ltd.)

Glycine decarboxylase (GLDC) acts in the glycine cleavage system to decarboxylate glycine and transfer a one-carbon unit into folate one-carbon metabolism. GLDC mutations cause a rare recessive disease non-ketotic hyperglycinemia (NKH). Mutations have also been identified in patients with neural tube defects (NTDs); however, the relationship between NKH and NTDs is unclear. We show that reduced expression of Gldc in mice suppresses glycine cleavage system activity and causes two distinct disease phenotypes. Mutant embryos develop partially penetrant NTDs while surviving mice exhibit post-natal features of NKH including glycine accumulation, early lethality and hydrocephalus. In addition to elevated glycine, Gldc disruption also results in abnormal tissue folate profiles, with depletion of one-carbon-carrying folates, as well as growth retardation and reduced cellular proliferation. Formate treatment normalizes the folate profile, restores embryonic growth and prevents NTDs, suggesting that Gldc deficiency causes NTDs through limiting supply of one-carbon units from mitochondrial folate metabolism.

Background: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions., Objective: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism., Design: Folic acid-supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr(+/+) and Mthfr(+/-) mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined., Results: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr(+/-) mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr(+/-) livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr(+/-) mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile., Conclusions: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.

The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the folate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation., (© FASEB.)

Purpose: Advanced methodologies for visualizing novel tissue contrast are essential for phenotyping the ever-increasing number of mutant mouse embryos being generated. Although diffusion microscopic MRI (μMRI) has been used to phenotype embryos, widespread routine use is limited by extended scanning times, and there is no established experimental procedure ensuring optimal data acquisition., Methods: We developed two protocols for designing experimental procedures for diffusion μMRI of mouse embryos, which take into account the effect of embryo preparation and pulse sequence parameters on resulting data. We applied our protocols to an investigation of the splotch mouse model as an example implementation., Results: The protocols provide DTI data in 24 min per direction at 75 μm isotropic using a three-dimensional fast spin-echo sequence, enabling preliminary imaging in 3 h (6 directions plus one unweighted measurement), or detailed imaging in 9 h (42 directions plus six unweighted measurements). Application to the splotch model enabled assessment of spinal cord pathology., Conclusion: We present guidelines for designing diffusion μMRI experiments, which may be adapted for different studies and research facilities. As they are suitable for routine use and may be readily implemented, we hope they will be adopted by the phenotyping community., (© 2014 Wiley Periodicals, Inc.)

Neural tube defects (NTDs) are among the commonest and most severe forms of developmental defect, characterized by disruption of the early embryonic events of central nervous system formation. NTDs have long been known to exhibit a strong genetic dependence, yet the identity of the genetic determinants remains largely undiscovered. Initiation of neural tube closure is disrupted in mice homozygous for mutations in planar cell polarity (PCP) pathway genes, providing a strong link between NTDs and PCP signaling. Recently, missense gene variants have been identified in PCP genes in humans with NTDs, although the range of phenotypes is greater than in the mouse mutants. In addition, the sequence variants detected in affected humans are heterozygous, and can often be detected in unaffected individuals. It has been suggested that interactions between multiple heterozygous gene mutations cause the NTDs in humans. To determine the phenotypes produced in double heterozygotes, we bred mice with all three pairwise combinations of Vangl2(Lp), Scrib(Crc) and Celsr1(Crsh) mutations, the most intensively studied PCP mutants. The majority of double-mutant embryos had open NTDs, with the range of phenotypes including anencephaly and spina bifida, therefore reflecting the defects observed in humans. Strikingly, even on a uniform genetic background, variability in the penetrance and severity of the mutant phenotypes was observed between the different double-heterozygote combinations. Phenotypically, Celsr1(Crsh);Vangl2(Lp);Scrib(Crc) triply heterozygous mutants were no more severe than doubly heterozygous or singly homozygous mutants. We propose that some of the variation between double-mutant phenotypes could be attributed to the nature of the protein disruption in each allele: whereas Scrib(Crc) is a null mutant and produces no Scrib protein, Celsr1(Crsh) and Vangl2(Lp) homozygotes both express mutant proteins, consistent with dominant effects. The variable outcomes of these genetic interactions are of direct relevance to human patients and emphasize the importance of performing comprehensive genetic screens in humans., (© 2014. Published by The Company of Biologists Ltd.)

The role of planar cell polarity (PCP) signalling in neural crest (NC) development is unclear. The PCP dependence of NC cell migration has been reported in Xenopus and zebrafish, but NC migration has not been studied in mammalian PCP mutants. Vangl2(Lp/Lp) mouse embryos lack PCP signalling and undergo almost complete failure of neural tube closure. Here we show, however, that NC specification, migration and derivative formation occur normally in Vangl2(Lp/Lp) embryos. The gene family member Vangl1 was not expressed in NC nor ectopically expressed in Vangl2(Lp/Lp) embryos, and doubly homozygous Vangl1/Vangl2 mutants exhibited normal NC migration. Acute downregulation of Vangl2 in the NC lineage did not prevent NC migration. In vitro, Vangl2(Lp/Lp) neural tube explants generated emigrating NC cells, as in wild type. Hence, PCP signalling is not essential for NC migration in mammals, in contrast to its essential role in neural tube closure. PCP mutations are thus unlikely to mediate NC-related birth defects in humans., (© 2014. Published by The Company of Biologists Ltd.)

Neural tube defects (NTDs), including spina bifida and anencephaly, are severe birth defects of the central nervous system that originate during embryonic development when the neural tube fails to close completely. Human NTDs are multifactorial, with contributions from both genetic and environmental factors. The genetic basis is not yet well understood, but several nongenetic risk factors have been identified as have possibilities for prevention by maternal folic acid supplementation. Mechanisms underlying neural tube closure and NTDs may be informed by experimental models, which have revealed numerous genes whose abnormal function causes NTDs and have provided details of critical cellular and morphological events whose regulation is essential for closure. Such models also provide an opportunity to investigate potential risk factors and to develop novel preventive therapies.

The immense challenge of annotating the entire mouse genome has stimulated the development of cutting-edge imaging technologies in a drive for novel information. These techniques promise to improve understanding of the genes involved in embryo development, at least one third of which have been shown to be essential. Aligning advanced imaging technologies with biological needs will be fundamental to maximising the number of phenotypes discovered in the coming years. International efforts are underway to meet this challenge through an integrated and sophisticated approach to embryo phenotyping. We review rapid advances made in the imaging field over the past decade and provide a comprehensive examination of the relative merits of current and emerging techniques. The aim of this review is to provide a guide to state-of-the-art embryo imaging that will enable informed decisions as to which technology to use and fuel conversations between expert imaging laboratories, researchers, and core mouse production facilities., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Rodent models of abdominal wall defects (AWD) may provide insight into the pathophysiology of these conditions including gut dysfunction in gastroschisis, or pulmonary hypoplasia in exomphalos. Previously, a Scribble mutant mouse model (circletail) was reported to exhibit gastroschisis. We further characterise this AWD in Scribble knockout mice., Method: Homozygous Scrib knockout mice were obtained from heterozygote matings. Fetuses were collected at E17.5-18.5 with intact amniotic membranes. Three mutants and two control fetuses were imaged by in amnio micro-MRI. Remaining fetuses were dissected, photographed and gut length/weight measured. Ileal specimens were stained for interstitial cells of Cajal (ICC), imaged using confocal microscopy and ICC quantified., Results: 127 fetuses were collected, 15 (12%) exhibited AWD. Microdissection revealed 3 mutants had characteristic exomphalos phenotype with membrane-covered gut/liver herniation into the umbilical cord. A further 12 exhibited extensive AWD, with eviscerated abdominal organs and thin covering membrane (intact or ruptured). Micro-MRI confirmed these phenotypes. Gut was shorter and heavier in AWD group compared to controls but morphology/number of ICC was not different., Discussion: The Scribble knockout fetus exhibits exomphalos (intact and ruptured), in contrast to the original published phenotype of gastroschisis. Detailed dissection of fetuses is essential ensuring accurate phenotyping and result reporting., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Genetic variants in one-carbon folate metabolism have been identified as risk factors for disease because they may impair the production or use of one-carbon folates required for nucleotide synthesis and methylation. p.R653Q (1958G>A) is a single-nucleotide polymorphism (SNP) in the 10-formyltetrahydrofolate (formylTHF) synthetase domain of the trifunctional enzyme MTHFD1; this domain produces the formylTHF which is required for the de novo synthesis of purines. Approximately 20% of Caucasians are homozygous for the Q allele. MTHFD1 p.R653Q has been proposed as a risk factor for neural tube defects (NTDs), congenital heart defects (CHDs) and pregnancy losses. We have generated a novel mouse model in which the MTHFD1 synthetase activity is inactivated without affecting protein expression or the other activities of this enzyme. Complete loss of synthetase activity (Mthfd1S(-/-)) is incompatible with life; embryos die shortly after 10.5 days gestation, and are developmentally delayed or abnormal. The proportion of 10-formylTHF in the plasma and liver of Mthfd1S(+/-) mice is reduced (P < 0.05), and de novo purine synthesis is impaired in Mthfd1S(+/-) mouse embryonic fibroblasts (MEFs, P < 0.005). Female Mthfd1S(+/-) mice had decreased neutrophil counts (P < 0.05) during pregnancy and increased incidence of developmental defects in embryos (P = 0.052). These findings suggest that synthetase deficiency may lead to pregnancy complications through decreased purine synthesis and reduced cellular proliferation. Additional investigation of the impact of synthetase polymorphisms on human pregnancy is warranted.

Closure of the neural tube during embryogenesis is a crucial step in development of the central nervous system. Failure of this process results in neural tube defects, including spina bifida and anencephaly, which are among the most common birth defects worldwide. Maternal use of folic acid supplements reduces risk of neural tube defects but a proportion of cases are not preventable. Folic acid is thought to act through folate one-carbon metabolism, which transfers one-carbon units for methylation reactions and nucleotide biosynthesis. Hence suboptimal performance of the intervening reactions could limit the efficacy of folic acid. We hypothesized that direct supplementation with nucleotides, downstream of folate metabolism, has the potential to support neural tube closure. Therefore, in a mouse model that exhibits folic acid-resistant neural tube defects, we tested the effect of specific combinations of pyrimidine and purine nucleotide precursors and observed a significant protective effect. Labelling in whole embryo culture showed that nucleotides are taken up by the neurulating embryo and incorporated into genomic DNA. Furthermore, the mitotic index was elevated in neural folds and hindgut of treated embryos, consistent with a proposed mechanism of neural tube defect prevention through stimulation of cellular proliferation. These findings may provide an impetus for future investigations of supplemental nucleotides as a means to prevent a greater proportion of human neural tube defects than can be achieved by folic acid alone.

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Neural tube defects are severe congenital malformations affecting around one in every 1000 pregnancies. An innovation in clinical management has come from the finding that closure of open spina bifida lesions in utero can diminish neurological dysfunction in children. Primary prevention with folic acid has been enhanced through introduction of mandatory food fortification in some countries, although not yet in the UK. Genetic predisposition accounts for most of the risk of neural tube defects, and genes that regulate folate one-carbon metabolism and planar cell polarity have been strongly implicated. The sequence of human neural tube closure events remains controversial, but studies of mouse models of neural tube defects show that anencephaly, open spina bifida, and craniorachischisis result from failure of primary neurulation, whereas skin-covered spinal dysraphism results from defective secondary neurulation. Other malformations, such as encephalocele, are likely to be postneurulation disorders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Folates act as co-factors for transfer of one-carbon units for nucleotide production, methylation and other biosynthetic reactions. Comprehensive profiling of multiple folates can be achieved using liquid chromatography tandem mass spectrometry, enabling determination of their relative abundance that may provide an indication of metabolic differences between cell types. For example, cell lines exposed to methotrexate showed a dose-dependent elevation of dihydrofolate, consistent with inhibition of dihydrofolate reductase. We analysed the folate profile of E. coli sub-types as well as cell lines and embryonic tissue from both human and mouse. The folate profile of bacteria differed markedly from those of all the mammalian samples, most notably in the greater abundance of formyl tetrahydrofolate. The overall profiles of mouse and human fibroblasts and mid-gestation mouse embryos were broadly similar, with specific differences. The major folate species in these cell types was 5-methyl tetrahydrofolate, in contrast to lymphoblastoid cell lines in which the predominant form was tetrahydrofolate. Analysis of embryonic human brain revealed a shift in folate profile with increasing developmental stage, with a decline in relative abundance of dihydrofolate and increase in 5-methyl tetrahydrofolate. These cell type-specific and developmental changes in folate profile may indicate differential requirements for the various outputs of folate metabolism.

Background: Lamins are intermediate filament proteins that form a major component of the nuclear lamina, a protein complex at the surface of the inner nuclear membrane. Numerous clinically diverse conditions, termed laminopathies, have been found to result from mutation of LMNA. In contrast, coding or loss of function mutations of LMNB1, encoding lamin B1, have not been identified in human disease. In mice, polymorphism in Lmnb1 has been shown to modify risk of neural tube defects (NTDs), malformations of the central nervous system that result from incomplete closure of the neural folds., Methods: Mutation analysis by DNA sequencing was performed on all exons of LMNB1 in 239 samples from patients with NTDs from the United Kingdom, Sweden, and United States. Possible functional effects of missense variants were analyzed by bioinformatics prediction and fluorescence in photobleaching., Results: In NTD patients, we identified two unique missense variants that were predicted to disrupt protein structure/function and represent putative contributory mutations. Fluorescence loss in photobleaching analysis showed that the A436T variant compromised stability of lamin B1 interaction within the lamina., Conclusion: The genetic basis of human NTDs appears highly heterogenous with possible involvement of multiple predisposing genes. We hypothesize that rare variants of LMNB1 may contribute to susceptibility to NTDs., (Copyright © 2013 The Authors. Birth Defects Research Part A published by Wiley-Blackwell.)

Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data.

The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy-and also to its side effects, which include folate deficiency and gastrointestinal upset., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Neural tube defects (NTDs) are severe congenital malformations affecting 1 in every 1000 pregnancies. 'Open' NTDs result from failure of primary neurulation as seen in anencephaly, myelomeningocele (open spina bifida), and craniorachischisis. Degeneration of the persistently open neural tube in utero leads to loss of neurological function below the lesion level. 'Closed' NTDs are skin-covered disorders of spinal cord structure, ranging from asymptomatic spina bifida occulta to severe spinal cord tethering, and usually traceable to disruption of secondary neurulation. 'Herniation' NTDs are those in which meninges, with or without brain or spinal cord tissue, become exteriorized through a pathological opening in the skull or vertebral column (e.g., encephalocele and meningocele). NTDs have multifactorial etiology, with genes and environmental factors interacting to determine individual risk of malformation. While over 200 mutant genes cause open NTDs in mice, much less is known about the genetic causation of human NTDs. Recent evidence has implicated genes of the planar cell polarity signaling pathway in a proportion of cases. The embryonic development of NTDs is complex, with diverse cellular and molecular mechanisms operating at different levels of the body axis. Molecular regulatory events include the bone morphogenetic protein and Sonic hedgehog pathways which have been implicated in control of neural plate bending. Primary prevention of NTDs has been implemented clinically following the demonstration that folic acid (FA), when taken as a periconceptional supplement, can prevent many cases. Not all NTDs respond to FA, however, and adjunct therapies are required for prevention of this FA-resistant category., (Copyright © 2012 Wiley Periodicals, Inc.)

Adhesion and fusion of epithelial sheets marks the completion of many morphogenetic events during embryogenesis. Neural tube closure involves an epithelial fusion sequence in which the apposing neural folds adhere initially via cellular protrusions, proceed to a more stable union, and subsequently undergo remodeling of the epithelial structures to yield a separate neural tube roof plate and overlying nonneural ectoderm. Cellular protrusions comprise lamellipodia and filopodia, and studies in several different systems emphasize the critical role of RhoGTPases in their regulation. How epithelia establish initial adhesion is poorly understood but, in neurulation, may involve interactions between EphA receptors and their ephrinA ligands. Epithelial remodeling is spatially and temporally correlated with apoptosis in the dorsal neural tube midline, but experimental inhibition of this cell death does not prevent fusion and remodeling. A variety of molecular signaling systems have been implicated in the late events of morphogenesis, but genetic redundancy, for example among the integrins and laminins, makes identification of the critical players challenging. An improved understanding of epithelial fusion can provide insights into normal developmental processes and may also indicate the mode of origin of clinically important birth defects., (Copyright © 2012 Wiley Periodicals, Inc.)

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein-protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism., (© 2011 Wiley Periodicals, Inc.)

Mutations have been identified in a non-canonical Wnt signalling cascade (the planar cell polarity pathway) in several mouse genetic models of severe neural tube defects. In each of these models, neurulation fails to be initiated at the 3-4 somite stage, leading to an almost entirely open neural tube (termed craniorachischisis). Studies in whole embryo culture have identified a defect in the morphogenetic process of convergent extension during gastrulation, preceding the onset of neural tube closure. The principal defect is a failure of midline extension, both in the neural plate and axial mesoderm. This leads to an abnormally wide neural plate in which the elevating neural folds are too far apart to achieve closure. In this chapter, we provide details of several experimental methods that can be used to evaluate convergent extension in cultured mouse embryos. We describe analytical methods that can reveal the abnormalities that characterise neurulation-stage embryos with defective planar cell polarity signalling, in particular the loop-tail (Lp; Vangl2) mutant.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects whose complex multigenic causation has hampered efforts to delineate their molecular basis. The effect of putative modifier genes in determining NTD susceptibility may be investigated in mouse models, particularly those that display partial penetrance such as curly tail, a strain in which NTDs result from a hypomorphic allele of the grainyhead-like-3 gene. Through proteomic analysis, we found that the curly tail genetic background harbours a polymorphic variant of lamin B1, lacking one of a series of nine glutamic acid residues. Lamins are intermediate filament proteins of the nuclear lamina with multiple functions that influence nuclear structure, cell cycle properties, and transcriptional regulation. Fluorescence loss in photobleaching showed that the variant lamin B1 exhibited reduced stability in the nuclear lamina. Genetic analysis demonstrated that the variant also affects neural tube closure: the frequency of spina bifida and anencephaly was reduced three-fold when wild-type lamin B1 was bred into the curly tail strain background. Cultured fibroblasts expressing variant lamin B1 show significantly increased nuclear dysmorphology and diminished proliferative capacity, as well as premature senescence, associated with reduced expression of cyclins and Smc2, and increased expression of p16. The cellular basis of spinal NTDs in curly tail embryos involves a proliferation defect localised to the hindgut epithelium, and S-phase progression was diminished in the hindgut of embryos expressing variant lamin B1. These observations indicate a mechanistic link between altered lamin B1 function, exacerbation of the Grhl3-mediated cell proliferation defect, and enhanced susceptibility to NTDs. We conclude that lamin B1 is a modifier gene of major effect for NTDs resulting from loss of Grhl3 function, a role that is likely mediated via the key function of lamin B1 in maintaining integrity of the nuclear envelope and ensuring normal cell cycle progression., Competing Interests: The authors have declared that no competing interests exist.

Myo-inositol plays key physiological functions, necessitating development of methodology for quantification in biological matrices. Limitations of current mass spectrometry-based approaches include the need for a derivatisation step and/or sample clean-up. In addition, co-elution of glucose may cause ion suppression of myo-inositol signals, for example in blood or urine samples. We describe an HPLC-MS/MS method using a lead-form resin based column online to a triple quadrupole tandem mass spectrometer, which requires minimum sample preparation and no derivatisation. This method allows separation and selective detection of myo-inositol from other inositol stereoisomers. Importantly, inositol was also separated from hexose monosaccharides of the same molecular weight, including glucose, galactose, mannose and fructose. The inter- and intra-assay variability was determined for standard solutions and urine with inter-assay coefficient of variation (CV) of 1.1% and 3.5% respectively, while intra-assay CV was 2.3% and 3.6%. Urine and blood samples from normal individuals were analysed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The molecular requirements for neural tube closure are complex. This is illustrated by the occurrence of neural tube defects (NTDs) in many genetic mouse mutants, which implicate a variety of genes, pathways and cellular functions. NTDs are also prevalent birth defects in humans, affecting around 1 per 1000 pregnancies worldwide. In humans the causation is thought to involve the interplay of fetal genes and the effect of environmental factors. Recent studies on the aetiology of human NTDs, as well as analysis of mouse models, have raised the question of the possible involvement of epigenetic factors in determining susceptibility. A consideration of potential causative factors in human NTDs must now include both alterations in the regulation of gene expression, through mutation of promoter or regulatory elements, and the additional analysis of epigenetic regulation. Alterations in the epigenetic status can be directly modified by various environmental insults or maternal dietary factors.

Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals., (Copyright © 2011 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Cranial neural tube defects (NTDs) occur in mice carrying mutant alleles of many different genes, whereas isolated spinal NTDs (spina bifida) occur in fewer models, despite being common human birth defects. Spina bifida occurs at high frequency in the Axial defects (Axd) mouse mutant but the causative gene is not known. In the current study, the Axd mutation was mapped by linkage analysis. Within the critical genomic region, sequencing did not reveal a coding mutation whereas expression analysis demonstrated significant up-regulation of grainyhead-like 2 (Grhl2) in Axd mutant embryos. Expression of other candidate genes did not differ between genotypes. In order to test the hypothesis that over-expression of Grhl2 causes Axd NTDs, we performed a genetic cross to reduce Grhl2 function in Axd heterozygotes. Grhl2 loss of function mutant mice were generated and displayed both cranial and spinal NTDs. Compound heterozygotes carrying both loss (Grhl2 null) and putative gain of function (Axd) alleles exhibited normalization of spinal neural tube closure compared with Axd/+ littermates, which exhibit delayed closure. Grhl2 is expressed in the surface ectoderm and hindgut endoderm in the spinal region, overlapping with grainyhead-like 3 (Grhl3). Axd mutants display delayed eyelid closure, as reported in Grhl3 null embryos. Moreover, Axd mutant embryos exhibited increased ventral curvature of the spinal region and reduced proliferation in the hindgut, reminiscent of curly tail embryos, which carry a hypomorphic allele of Grhl3. Overall, our data suggest that defects in Axd mutant embryos result from over-expression of Grhl2.

Ambitious international efforts are underway to produce gene-knockout mice for each of the 25,000 mouse genes, providing a new platform to study mammalian development and disease. Robust, large-scale methods for morphological assessment of prenatal mice will be essential to this work. Embryo phenotyping currently relies on histological techniques but these are not well suited to large volume screening. The qualitative nature of these approaches also limits the potential for detailed group analysis. Advances in non-invasive imaging techniques such as magnetic resonance imaging (MRI) may surmount these barriers. We present a high-throughput approach to generate detailed virtual histology of the whole embryo, combined with the novel use of a whole-embryo atlas for automated phenotypic assessment. Using individual 3D embryo MRI histology, we identified new pituitary phenotypes in Hesx1 mutant mice. Subsequently, we used advanced computational techniques to produce a whole-body embryo atlas from 6 CD-1 embryos, creating an average image with greatly enhanced anatomical detail, particularly in CNS structures. This methodology enabled unsupervised assessment of morphological differences between CD-1 embryos and Chd7 knockout mice (n=5 Chd7(+/+) and n=8 Chd7(+/-), C57BL/6 background). Using a new atlas generated from these three groups, quantitative organ volumes were automatically measured. We demonstrated a difference in mean brain volumes between Chd7(+/+) and Chd7(+/-) mice (42.0 vs. 39.1mm(3), p<0.05). Differences in whole-body, olfactory and normalised pituitary gland volumes were also found between CD-1 and Chd7(+/+) mice (C57BL/6 background). Our work demonstrates the feasibility of combining high-throughput embryo MRI with automated analysis techniques to distinguish novel mouse phenotypes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Folate one-carbon metabolism has been implicated as a determinant of susceptibility to neural tube defects (NTDs), owing to the preventive effect of maternal folic acid supplementation and the higher risk associated with markers of diminished folate status., Methods: Folate one-carbon metabolism was compared in curly tail (ct/ct) and genetically matched congenic (+(ct)/+(ct)) mouse strains using the deoxyuridine suppression test in embryonic fibroblast cells and by quantifying s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) in embryos using liquid chromatography tandem mass spectrometry. A possible genetic interaction between curly tail and a null allele of 5,10-methylenetetrahydrofolate reductase (MTHFR) was investigated by generation of compound mutant embryos., Results: There was no deficit in thymidylate biosynthesis in ct/ct cells, but incorporation of exogenous thymidine was lower than in +(ct)/+(ct) cells. In +(ct)/+(ct) embryos the SAM/SAH ratio was diminished by dietary folate deficiency and normalized by folic acid or myo-inositol treatment, in association with prevention of NTDs. In contrast, folate deficiency caused a significant increase in the SAM/SAH ratio in ct/ct embryos. Loss of MTHFR function in curly tail embryos significantly reduced the SAM/SAH ratio but did not cause cranial NTDs or alter the frequency of caudal NTDs., Conclusions: Curly tail fibroblasts and embryos, in which Grhl3 expression is reduced, display alterations in one-carbon metabolism, particularly in the response to folate deficiency, compared to genetically matched congenic controls in which Grhl3 is unaffected. However, unlike folate deficiency, diminished methylation potential appears to be insufficient to cause cranial NTDs in the curly tail strain, nor does it increase the frequency of caudal NTDs.

It is becoming apparent that regulation at the protein level plays crucial roles in developmental and pathological processes. Therefore, we performed a proteomics screen to identify proteins that are differently expressed or modified at stages of development permissive (E11) and nonpermissive for regeneration (E15) of the chick spinal cord. Proteins regulated either developmentally or in response to spinal-cord injury included collapsin-response-mediator proteins (Crmps), known to modulate microtubule dynamic and axonal growth. No significant changes in Crmp transcripts following injury were observed, indicating regulation mainly at the protein level. Analysis of Crmp-2 protein and its phosphorylated forms, pS522 and pT514, showed that Crmp-2 is developmentally regulated and also expressed in neural progenitors in vivo and in neurospheres. Its cellular localization changed both with development and following spinal-cord injury. In addition, although overall levels of Crmp-2 expression were not affected by injury, abundance of certain phosphorylated forms was altered. pT514 Crmp-2 appeared to be associated with dividing neural progenitors and was greatly reduced at nonpermissive stages for regeneration, whereas it did not seem affected by injury. In contrast, phosphorylation of Crmp-2 at S522 was upregulated early after injury in regenerating spinal cords and the ratio between phosphorylated to total Crmp-2 increased, as indicated by 2D Western blots. Altogether, this study shows highly dynamic regulation of Crmp-2 forms during development and identifies post-translational changes in Crmp-2 as putative contributors to the maintenance of spinal-cord regenerative ability, possibly via a transient stabilization of the neuronal cytoskeleton.

Background: Suboptimal maternal folate status is considered a risk factor for neural tube defects (NTDs). However, the relationship between dietary folate status and risk of NTDs appears complex, as experimentally induced folate deficiency is insufficient to cause NTDs in nonmutant mice. In contrast, folate deficiency can exacerbate the effect of an NTD-causing mutation, as in splotch mice. The purpose of the present study was to determine whether folate deficiency can induce NTDs in mice with a permissive genetic background which do not normally exhibit defects., Methods: Folate deficiency was induced in curly tail and genetically matched wild-type mice, and we analyzed the effect on maternal folate status, embryonic growth and development, and frequency of NTDs., Results: Folate-deficient diets resulted in reduced maternal blood folate, elevated homocysteine, and a diminished embryonic folate content. Folate deficiency had a deleterious effect on reproductive success, resulting in smaller litter sizes and an increased rate of resorption. Notably, folate deficiency caused a similar-sized, statistically significant increase in the frequency of cranial NTDs among both curly tail (Grhl3 mutant) embryos and background-matched embryos that are wild type for Grhl3. The latter do not exhibit NTDs under normal dietary conditions. Maternal supplementation with myo-inositol reduced the incidence of NTDs in the folate-deficient wild-type strain., Conclusions: Dietary folate deficiency can induce cranial NTDs in nonmutant mice with a permissive genetic background, a situation that likely parallels gene-nutrient interactions in human NTDs. Our findings suggest that inositol supplementation may ameliorate NTDs resulting from insufficient dietary folate.

Mammalian neurulation is completed when the dorsolateral neural folds bend inwards, their tips make adhesive contacts across the midline, and the epithelia remodel to create a closed neural tube. Two recent papers (one by Camerer et al. in this issue of Developmental Cell) demonstrate a vital role for protease-activated G protein-coupled receptor signaling in these late closure events, opening up new avenues for exploring the molecular basis of mammalian neural tube morphogenesis., ((c) 2010 Elsevier Inc. All rights reserved.)