Search

The Transforming growth factor-β (TGF-β) family control diverse cellular processes and specify cell-fate/differentiation during embryogenesis in vertebrates and invertebrates. Mutations disrupting TGF-β signalling lead to developmental abnormalities and a range of diseases such as cancer. Nodal is a major TGF-β signal, responsible for gastrulation in embryogenesis. Arkadia (Akd) was discovered by mouse gene-trap mutagenesis and encodes a nuclear E3 ubiquitin ligase. Akd allows the Nodal signal to reach its maximum level and Akd-null mice lack mammalian organiser (MO) and mesendodermal tissues. Although Akd RNA is ubiquitously expressed, Akd-null mice lose a subset of Nodal-dependent functions. The specificity of Akd function is therefore most likely to be regulated post-transcriptionally or by co-factors. Akd possesses differentially spliced 5′ untranslated regions (UTRs) and large 3′ UTR. We have employed bioinformatics and developed a reporter system to address Akd post-transcriptional regulation. Akd RNA may initiate from different promoters and 5′ UTR differential splicing, upstream AUGs (uAUGs) and open-reading frames upstream (uORFs) may regulate protein translation. 5′ and 3′ UTRs can interact to either destabilise or decrease translational efficiency of RNA. The nature of this interaction is cell-type and signal level dependent. These data may represent mechanisms by which translational control of Arkadia is achieved and ultimately how TGF-β/Nodal signalling is regulated during embryogenesis.

Cranial neurulation is the embryonic process responsible for formation of the brain primordium. In the mouse embryo, cranial neurulation is a piecemeal process with several initiation sites and two neuropores. Variation in the pattern of cranial neurulation occurs in different mouse strains, and a simpler version of this morphogenetic scheme has been described in human embryos. Exencephaly is more common in females than in males, an unexplained phenomenon seen in both mice and humans. As the cranial neural tube closes, a critical morphogenetic event is the formation of dorsolateral bending points near the neural fold tips, which enables subsequent midline fusion of the neural folds. Many mutant and gene-targeted mouse strains develop cranial neural tube defects, and analysis of the underlying molecular defects identifies several requirements for normal dorsolateral bending. These include a functional actin cytoskeleton, emigration of the cranial neural crest, spatio-temporally regulated apoptosis, and a balance between cell proliferation and the onset of neuronal differentiation. A small number of mouse mutants exhibit craniorachischisis, a combined brain and spine neurulation defect. Recent studies show that disturbance of a single molecular signalling cascade, the planar cell polarity pathway, is implicated in mutants with this defect.

Loop-tail ( Lp ) mice develop double outlet right ventricle and aortic arch defects, and the defects are caused by mutations in the Vangl2 gene. Vangl2 mRNA is found in the outflow tract myocardium, including the myocardializing cells that migrate into the outflow tract cushions. Analysis of muscularization of the outflow tract septum showed that this process is compromised in Lp/Lp fetuses. Vangl2 is a component of the noncanonical Wnt, planar cell polarity (PCP) pathway that signals via RhoA. We therefore looked for evidence of polarization in myocardializing cells. In wild-type fetuses, myocardializing cells extend lamellipodia and filopodia into the cushion tissue and reorganize their actin cytoskeleton from a cortical form to stress fibers; behaviors that are characteristic of polarized cells. In contrast, Lp/Lp littermates do not extend lamellipodia or filopodia into the cushion tissue, and their actin remains in a cortical form, suggesting that polarized cell migration of myocardializing cells is inhibited in Lp/Lp . Several other components of the PCP pathway are also localized in the outflow tract myocardium. In wild-type fetuses, the myocardializing cells coexpress RhoA and one of its downstream mediators, ROCK1. RhoA expression is disrupted in Lp/Lp , and is lost from the myocardial-cushion tissue interface, including the presumptive myocardializing cells. These data suggest that Vangl2 is required for the polarization and movement of myocardializing cells into the outflow tract cushions, and that RhoA and ROCK1 are downstream mediators of the PCP signaling pathway in the developing outflow tract.

We generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein -- diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells.

A proportion of neural tube defects (NTDs) can be prevented by maternal folic acid supplementation, although some cases are unresponsive. The curly tail mutant mouse provides a model of folate-resistant NTDs, in which defects can be prevented by inositol therapy in early pregnancy. Hence, inositol represents a possible novel adjunct therapy to prevent human NTDs. The present study investigated the molecular mechanism by which inositol prevents mouse NTDs. Activation of protein kinase C (PKC) is known to be essential, and we examined neurulation-stage embryos for PKC expression and applied PKC inhibitors to curly tail embryos developing in culture. Although all known PKC isoforms were detected in the closing neural tube, use of chemical PKC inhibitors identified a particular requirement for 'conventional' PKC isoforms. Peptide inhibitors offer selective inhibition of individual PKCs, and we demonstrated isoform-specific inhibition of PKC in embryonic cell cultures. Application of peptide inhibitors to neurulation-stage embryos revealed an absolute dependence on the activity of PKCbetaI and gamma for prevention of NTDs by inositol, and partial dependence on PKCzeta, whereas other PKCs (alpha, betaII delta, and epsilon) were dispensable. To investigate the cellular action of inositol and PKCs in NTD prevention, we examined cell proliferation in curly tail embryos. Defective proliferation of hindgut cells is a key component of the pathogenic sequence leading to NTDs in curly tail. Hindgut cell proliferation was stimulated specifically by inositol, an effect that required activation of PKCbetaI. Our findings reveal an essential role of specific PKC isoforms in mediating the prevention of mouse NTDs by inositol.

More than 80 mutant mouse genes disrupt neurulation and allow an in-depth analysis of the underlying developmental mechanisms. Although many of the genetic mutants have been studied in only rudimentary detail, several molecular pathways can already be identified as crucial for normal neurulation. These include the planar cell-polarity pathway, which is required for the initiation of neural tube closure, and the sonic hedgehog signalling pathway that regulates neural plate bending. Mutant mice also offer an opportunity to unravel the mechanisms by which folic acid prevents neural tube defects, and to develop new therapies for folate-resistant defects.

We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA[2, 3]. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.

The observation of elevated maternal plasma homocysteine concentration in pregnancies affected by neural tube defects (NTD) suggests that folate metabolism may be disturbed in NTD cases. In addition, studies on the chick embryo suggest that hyperhomocysteinaemia may contribute directly to the development of NTD. In order to test the hypothesis that homocysteine may cause NTD, we cultured mouse embryos in the presence of homocysteine thiolactone during the period of cranial neural tube closure. At doses of 0.5 mM or above, exposure to homocysteine thiolactone caused growth retardation, blisters and abnormalities of somite development. Despite the teratogenic effects of homocysteine we did not detect any increase in the incidence of neural tube defects. Neither was there an effect on the incorporation of thymidine into DNA, a potential marker of alterations in the folate or homocysteine/methionine cycles. These observations suggest that homocysteine is unlikely to be a direct cause of NTD in humans. Rather, the elevated levels of homocysteine in human NTD pregnancies may reflect a disturbance in folate-related metabolism.

During early embryonic development, cell migration is one of the most important morphogenetic processes. Neural crest cells arise from the dorsal part of the neural tube and migrate along different pathways to numerous locations where they differentiate into a variety of tissues. In the mouse, studies of neural crest cell migration have been difficult partly because of the absence of specific markers which can label neural crest cells throughout their migration from their origin to the site of differentiation. Nevertheless, the use of different experimental strategies involving extrinsic, intrinsic or genetic cell markers has already led to a good understanding of this migration. In our studies, extrinsic markers such as wheat germ agglutinin-gold conjugates and DiI and genetic markers including Hoxb2-lacZ and green fluorescent protein have been employed in tracing migrating neural crest cells. The labelling procedures and the strength and weaknesses of the tracing methods are reviewed herein.

Ahnak is a gigantic (700 kD) phosphoprotein with a unique structure whose expression and cellular localization are dynamically regulated during cell cycle progression. Here, we report that Ahnak is localized to sites of major morphogenesis during mouse placentation and neurulation. Ahnak was found in: (i) derivatives of trophectoderm, including chorionic ectoderm prior to and during union with the ectoplacental cone, presumptive syncytiotrophoblast cells in the chorionic labyrinth, and giant cells at the trophoblast-uterine interface; (ii) the allantois prior to, during, and after union with the chorion; and (iii) the tips of the neural plate during formation of the neural tube. On the basis of these observations, we suggest that Ahnak may play heretofore unrecognized roles in tissue union during normal mouse development.

Sponsored by: The Genetics Society, Promega (UK) Ltd and B&K Universal Group Ltd

Heparan sulphate proteoglycans have been implicated in the binding and presentation of several growth factors to their receptors, thereby regulating cellular growth and differentiation. To investigate the role of heparan sulphate proteoglycans in mouse spinal neurulation, we administered chlorate, a competitive inhibitor of glycosaminoglycan sulphation, to cultured E8.5 embryos. Treated embryos exhibit accelerated posterior neuropore closure, accompanied by suppression of neuroepithelial bending at the median hinge point and accentuated bending at the paired dorsolateral hinge points of the posterior neuropore. These effects appear specific, as they can be prevented by addition of heparan sulphate to the culture medium, whereas heparitinase-treated heparan sulphate and chondroitin sulphate are ineffective. Both N- and O-sulphate groups appear to be necessary for the action of heparan sulphate. In situ hybridisation analysis demonstrates a normal distribution of sonic hedgehog mRNA in chlorate-treated embryos. By contrast, patched 1 transcripts are abnormally abundant in the notochord, and diminished in the overlying neuroepithelium, suggesting that sonic hedgehog signalling from the notochord may be perturbed by inhibition of heparan sulphation. Together, these results demonstrate a regulatory role for heparan sulphate in mouse spinal neurulation.

Normal growth and morphogenesis of the cranial vault reflect a balance between cell proliferation in the sutures and osteogenesis at the margins of the cranial bones. In the clinical condition craniosynostosis, the sutures fuse prematurely as a result of precocious osteogenic differentiation and craniofacial malformation results. Mutations in several fibroblast growth factor receptor (FGFR) genes have now been identified as being responsible for the major craniosynostotic syndromes. We have used a grafting technique to manipulate the levels of endogenous FGF-2 ligand in embryonic chick cranial vaults and thereby perturb morphogenesis. Implantation of beads loaded with FGF-2 did not affect normal cranial development at physiological concentrations, although they elicited a morphogenetic response in the limb. Implantation of beads loaded with a neutralising antibody to FGF-2 generated a concentration-dependent response. When a single bead was implanted, the grafts grew to a massive size as a result of increased cell division in the tissue. With greater inactivation of FGF-2 protein (two to three beads implanted), all further bone differentiation and cell proliferation was blocked. These data further support the emerging idea that the intensity of FGF-mediated signalling determines the developmental fate of the skeletogenic cells in the cranial vault. High and low levels correlate with differentiation and proliferation, respectively. A balance between the two ensures normal cranial vault morphogenesis. This is consistent with the observation that several FGFR mutations causing craniosynostosis result in constitutive activation of the receptor.

Cited2 (also Mrg1/p35srj) is a member of a new conserved gene family that is expressed during mouse development and in adult tissues. In order to investigate the function of Cited2 during mouse embryogenesis, we introduced a null mutation into the Cited2 locus. Cited2(-/-) mutants died at late gestation and exhibited heart defects and exencephaly, arising from defective closure of the midbrain (MB) and hindbrain. Initiation of neural tube closure at the forebrain-midbrain (FB-MB) boundary, an essential step for closure of the cranial neural tube, was impaired in the Cited2(-/-) mutants. Gene marker analysis using in situ hybridization revealed that the patterning of the anterior neural plate and head mesenchyme was little affected or normal in the Cited2(-/-) embryos. However, Cited2 was required for the survival of neuroepithelial cells and its absence led to massive apoptosis in dorsal neuroectoderm around the FB-MB boundary and in a restricted transverse domain in the hindbrain. Treatment with folic acid significantly reduced the exencephalic phenotype in the Cited2(-/-) embryos both in vivo and in vitro. However, assessment of folate metabolism revealed no defect in the Cited2(-/-) mutants, and the elevated apoptosis observed in the neuroepithelium of the Cited2(-/-) mutants was apparently not decreased by folic acid supplementation. To our knowledge, the Cited2 mouse represents the first genetic model in which folic acid can prevent a defect in neural tube closure by a mechanism other than the neutralization of a defect in folate homeostasis.

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., Summary: Actomyosin assembly and myosin ATPase activity are not essential for mouse spinal neurulation. However, the ROCK–LIMK–cofilin pathway, which controls actin turnover and actomyosin disassembly, is necessary.

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 human patients 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 human patients. To determine the phenotypes produced in double heterozygotes we bred mice with all three pairwise combinations of Vangl2Lp, ScribCrc and Celsr1Crsh 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, Celsr1Crsh; Vangl2Lp; ScribCrc 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 may be attributed to the nature of the protein disruption in each allele: while ScribCrc is a null mutant and produces no Scrib protein, Celsr1Crsh and Vangl2Lp 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.

The homozygous loop-tail ( Lp ) mouse has a severe neural tube closure defect, analogous to the craniorachischisis phenotype seen in humans. Linkage analysis and physical mapping have previously localized the Lp locus to a region on mouse chromosome 1 defined by the markers D1Mit113–Tagln2. Here we report the construction of sequence-ready bacterial clone contigs encompassing the Lp critical region in both mouse and the orthologous human region (1q22–q23). Twenty-two genes, one EST, and one pseudogene have been identified using a combination of EST database screening, exon amplification, and genomic sequence analysis. The preliminary gene map is Cen– Estm33 –AA693056– Ly9–Cd48–Slam–Cd84–Kiaa1215–Nhlh1–Kiaa0253–Copa–Pxf–H326–Pea15–Casq1–Atp1a4–Atp1a2–Estm34–Kcnj9–Kcnj10–Kiaa1355–Tagln2–Nesg1–Crp –Tel. The genes between Slam and Kiaa1355 are positional candidates for Lp. The comparative gene content and order are identical between mouse and human, indicating a high degree of conservation between the two species in this region. Together, the physical and transcript maps described here serve as resources for the identification of the Lp mutation and further define the conservation of this genomic region between mouse and human.

Sponsored by: The Genetical Society, Promega (UK) Ltd and B&K Universal Group Ltd

RhoB mRNA expression was examined in the developing mouse embryo between E8.5 and E11.5. Specific expression was found in migrating neural crest (NC) cells, from the first stages of their migration at E9.5, throughout the migration period. Expression is maintained in NC derivatives for at least one embryonic day after they reach their final destinations, but is then down-regulated. RhoB is also expressed in non NC-derived neural tissues, including motor neurones and the floor plate of the neural tube. RhoB mRNA expression is also found in the developing endocardial cushions of the atrioventricular and outflow regions of the developing heart.

Genetic polymorphisms are thought to play an important role in determining susceptibility to neural tube defects (NTDs), for example between different ethnic groups, but the embryonic manifestation of these polymorphic genetic influences is unclear. We have used a mouse model to test experimentally whether polymorphic variations in the pattern of cranial neural tube closure can influence susceptibility to NTDs. The site at which cranial neural tube closure begins (so-called closure 2) is polymorphic between inbred mice. Strains with a caudal location of closure 2 (e.g. DBA/2) are relatively resistant to NTDs, whereas strains with a rostrally positioned closure 2 (e.g. NZW) exhibit increased susceptibility to NTDs. We tested experimentally whether altering the position of closure 2 can affect susceptibility to cranial NTDs, by back- crossing the splotch ( Sp (2H) ) mutant gene onto the DBA/2 background. As a control, Sp (2H) was transferred onto the NZW background, which resembles splotch mice in its closure pattern. Approximately 80% of Sp (2H) homozygotes develop NTDs, both cranial (exencephaly) and spinal (spina bifida). After transfer to the DBA/2 background, the frequency of cranial NTDs was reduced significantly in Sp (2H) homozygotes, confirming a protective effect of caudal closure 2. In contrast, Sp (2H) homozygotes on the NZW background had a persistently high frequency of cranial NTDs. The frequency of spina bifida was not altered in either backcross, emphasizing the specificity of this genetic effect for cranial neurulation. These findings demonstrate that variation in the pattern of cranial neural tube closure is a genetically determined factor influencing susceptibility to cranial NTDs.

The spectrum of neuronal migration disorders (NMD) in humans encompasses developmental brain defects with a range of clinical and pathological features. A simple classification distinguishes agyria/pachygyria, heterotopia, polymicrogyria and cortical dysplasia as distinct clinico-pathological entities. Many of these conditions are associated with intractable epilepsy. When considering the pathogenesis of NMD, a critical developmental process is the migration of neuroblasts along the processes of radial glia during the formation of the layered structure of the cerebral cortex. In addition, faulty cytodifferentiation and programmed cell death play important roles in the generation of dysplasias and heterotopias respectively. A number of genes have been identified that participate in the regulation of neuronal migration. Mouse models, in which these genes are mutated, provide insight into the developmental pathways that underlie normal and abnormal neuronal migration.

Sponsored by: The Wellcome Trust and B & K Universal Group Limited

To examine the role of actin microfilaments in mouse spinal neurulation, we stained cryosections of E8.5-10.5 CBA/Ca embryos with FITC-phalloidin. Microfilaments are present in the apical region of all cells throughout the neuroepithelium, irrespective of whether they are involved in bending of the neural plate. Disruption of the microfilaments with cytochalasin D inhibited closure of the cranial neural folds in cultured embryos, even at the lowest concentrations tested, and prevented the initiation of spinal neurulation (Closure 1) at higher concentrations. In contrast, closure of the posterior neuropore was resistant to cytochalasin D at the highest concentrations tested. Phalloidin staining and transmission electron microscopy confirmed that cytochalasin D is effective in disassembling microfilaments in spinal neuroepithelial cells. We conclude that spinal neural tube closure does not require microfilament function, in contrast to cranial neurulation which is strongly microfilament-dependent. Histological examination of cytochalasin D-treated embryos revealed that bending at hinge points, both in the midline (MHP) and dorsolaterally (DLHPs), continues in the absence of microfilaments, whereas the rigidity of non-bending regions of the neural plate is lost. This suggests that spinal neurulation can continue in the presence of cytochalasin D largely as a result of intrinsic bending of the neural plate at hinge points. Cytochalasin D treatment is a useful tool for revealing the localisation of hinge points in the neural plate. Analysis of treated embryos demonstrates a transition, along the spinal axis, from closure solely involving midline bending, at high levels of the spinal axis, to closure solely involving dorsolateral bending, low in the spinal region. These findings support the idea of mechanistic heterogeneity in mouse neurulation, along the body axis, and demonstrate that contraction of actin microfilaments is not obligatory for epithelial bending during embryonic morphogenesis. Dev Dyn 1999;215:273-283.

The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis. To identify the Lp gene, a positional cloning approach has been adopted. Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1. Here we report a detailed physical map of this region. The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb. Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval. Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags. Added to other known genes in the region, a total of 29 genes were located and ordered within the contig. Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene. The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb. Within this region, 10 potential candidate genes have been mapped. The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q.

Nhlh1 is a basic helix-loop-helix (bHLH) gene that has been implicated in mouse neurogenesis. Previous studies have shown it to be expressed in regions in which there are differentiating neurons during late embryonic and fetal development, but detailed studies of the role of Nhlh1 earlier in embryonic development have not been performed. In this paper, we examine the expression of Nhlh1 transcripts at early embryonic stages (E8.5-E10.5), at the onset of neurogenesis, and compare the pattern of expression with that of Islet-1, a marker of postmitotic neurons. We show that Nhlh1 is expressed in early postmitotic neurons but is down-regulated as these cells migrate from the ventricular zone. We have also determined the genomic structure of mouse Nhlh1 and have characterised the promoter sequence, as a first step towards identifying factors that may control Nhlh1 expression. Nhlh1 has been implicated previously as a candidate for the neural tube defect mutant loop-tail (Lp); here, we present sequence and expression data indicating that Nhlh1 is unlikely to be responsible for the Lp mutation.

Arylamine N-acetyltransferases (NATs) are important in susceptibility to xenobiotic-induced disorders (e.g., drug-induced autoimmune disease, bladder cancer), but their role in endogenous metabolism is yet to be elucidated. The discovery that human NAT1 acts upon p-aminobenzoylgluatamate (p-ABG) to generate p-acetamidobenzoylglutamate (p-AABG), a major urinary metabolite of folic acid, suggests that human NAT1 may play a role in folic acid metabolism and hence in the normal development of the neural tube. In this study we examined the distribution of NAT in neuronal tissue from adult mice and embryos. Immunohistochemical staining of the adult mouse cerebellum revealed NAT2 (the mouse homologue of human NAT1) expression in the cell bodies and dendrites of Purkinje cells and in the neuroglia of the molecular layer. In embryos, NAT2 was detected in developing neuronal tissue on days 9.5, 11.5, and 13.5. It was expressed intensely in the nerual tube around the time of closure. The level of expression subsequently declined in the neuroepithelium but increased in glial cells. In addition, NAT2 was detected in the developing heart and gut. These findings demonstrate that the embryo itself expresses an enzyme which is involved in the metabolism of folic acid, so that the role played by both mother and embryo must be considered when examining the role of folic acid in embryonic development. These findings imply that polymorphisms in NAT genes could play a role in determining susceptibility to neural tube defects (NTD) and orofacial clefting, developmental disorders which can be prevented by dietary administration of folic acid.

Sponsored by: The Wellcome Trust, The Genetical Society and B&K Universal Group Limited

SM22alpha (TAGLN) is one of the earliest markers of differentiated smooth muscle, being expressed exclusively in the smooth muscle cells of adult tissues and transiently in embryonic skeletal and cardiac tissues. We have identified and mapped the mouse Tagln gene and a closely related gene, Sm22alpha homolog (Tagln2). The chromosomal localization for Tagln was identified by linkage analysis to distal mouse chromosome 9 between D9Mit154 and D9Mit330, closely linked to the anchor locus D9Nds10. The localization of Tagln2 was also determined and was found to map between Fcgr2 and D1Mit149 on distal mouse chromosome 1. This localization is homologous to a region of human 1q21-q25 to which an EST representing human TAGLN2 was previously mapped. The two regions, distal mouse chromosome 1 and proximal mouse chromosome 9, and the human regions with conserved synteny (1q21-q25 and 11q22-qter) are believed to be paralogous, reflecting either conserved remnants of duplicated chromosomes or segments of chromosomes during vertebrate evolution.

Neural tube defects can be prevented by folic acid, although the mechanism of this action is unclear. Studies of a series of folate-related enzymes have so far failed to pin-point the nature of the metabolic defect in the neurulation-stage embryo that is corrected by folic acid. Approximately 30% of neural tube defects appear resistant to folic acid and recent work in a mouse genetic model system suggests that administration of myo-inositol may be a complementary therapeutic option. The large number of mouse genes known to cause neural tube defects provide a starting point for identifying the genetic basis of the human defects. (C) 1998 Rapid Science Ltd.

Folic acid prevents up to 70% of human neural tube defects (NTD), offering the possibility of effective primary prevention for these crippling birth defects. Recent studies with mouse models of NTD aimed to shed light on two unsolved problems of NTD prevention: 1) the mechanism of action of folic acid, and 2) the development of an alternative therapy to prevent NTD that are unresponsive to folic acid. Using a biochemical screening test in cultured mouse embryos, we identified a defect of folate metabolism in embryos homozygous for the splotch (Sp(2H)) mutation that develop anencephaly and spina bifida. In contrast, there are no demonstrable abnormalities of folate metabolism in two other mouse mutants, curly tail and loop-tail, which also develop NTD. Exogenous folic acid prevents up to 50% of NTD in homozygous splotch embryos. Thymidine is also effective in preventing a proportion of NTD in this mutant, suggesting an inborn error in the provision of folate intermediates for pyrimidine biosynthesis in splotch mice. NTD in mutant curly tail mice do not respond to folic acid but are preventable in up to 70% of cases by exogenous myo-inositol. Embryo culture studies show this effect to be mediated via stimulation of protein kinase C and upregulation of retinoic acid receptor beta expression in the embryonic hindgut. Inositol therapy may represent a possible means of preventing folate-resistant NTD in humans. (C) 1998 Wiley-Liss, Inc.

Splotch mice, which harbour mutations in the Pax3 gene, exhibit neural crest-related abnormalities including pigmentation defects, reduced or absent dorsal root ganglia and failure of cardiac outflow tract septation in homozygotes. Although splotch neural crest cells fail to colonise target tissues, they initiate migration in vivo and appear to migrate as well as wild type neural crest cells in vitro, suggesting that the neural crest abnormality in splotch may reside not in the neural crest cells themselves, but rather in the extracellular environment through which they migrate. We have examined the expression of genes encoding extracellular matrix molecules in Sp 2H homozygous embryos and find a marked over-expression of transcripts for the chondroitin sulphate proteoglycan versican in the pathways of neural crest cell migration. Use of cadherin-6 expression as a marker for neural crest demonstrates a striking correlation between up-regulation of versican expression and absence of migrating neural crest cells, both in the mesenchyme lateral to the neural tube and in the lower branchial arches of Sp 2H homozygotes. Pax3 and versican have mutually exclusive expression patterns in normal embryos whereas, in Sp 2H homozygotes, versican is generally over-expressed with `infilling' in regions that would normally express functional Pax3 . Versican , like other chondroitin sulphate proteoglycans, is non-permissive for migration of neural crest cells in vitro, and we suggest that over-expression of this molecule leads to the arrest of neural crest cell migration in splotch embryos. Pax3 may serve to negatively regulate versican expression during normal development, thereby guiding neural crest cells into their pathways of migration.

Development of the neural crest involves a remarkable feat of coordinated cell migration in which cells detach from the neural tube, take varying routes of migration through the embryonic tissues and then differentiate at the end of their journey to participate in the formation of a number of organ systems. In general, neural crest cells appear to migrate without the guidance of long-range physical or chemical cues, but rather they respond to heterogeneity in the extracellular matrix that forms their migration substrate. Molecules such as fibronectin and laminin act as permissive substrate components, encouraging neural crest cell attachment and spreading, whereas chondroitin sulphate proteoglycans are nonpermissive for migration. A balance between permissive and nonpermissive substrate components seems to be necessary to ensure successful migration, as indicated by a number of studies in mouse mutant systems where nonpermissive molecules are over-expressed, leading to inhibition of neural crest migration. The neural crest expresses cell surface receptors that permit interaction with the extracellular matrix and may also modify the matrix by secretion of proteases. Thus the principles that govern the complex migration of neural crest cells are beginning to emerge.

Loop-tail (Lp) is unique among mouse mutants in failing to initiate neural tube closure at the cervical/hindbrain boundary (so-called 'Closure 1'), at the 5-7 somite stage. Lp/Lp embryos go on to develop a malformation that closely resembles cranio-rachischisis, the most severe neural tube defect found in humans. We investigated several possible embryological mechanisms that may underlie this failure of neural tube closure in Lp. The genotypes of Lp/Lp, Lp/+ and +/+ embryos from mixed litters were identified using the polymerase chain reaction to amplify a polymorphic microsatellite sequence that is very closely linked to Lp. At post-neurulation stages of development, Lp/Lp embryos have a shortened body axis, which could suggest a defect of axial elongation as the primary anomaly in Lp. However, we found that axial elongation is normal in Lp homozygotes prior to the stage of defective Closure 1, indicating that the shortened body axis of later embryos is a secondary effect of the neurulation anomaly, or an independent effect of the Lp mutation. Some workers have reported cell proliferation rates to be abnormal in later stage Lp/Lp embryos. We observed variations in [3H]thymidine labelling index, and mitotic index, between embryonic tissues, and between embryos at different somite stages. However, Lp/Lp, Lp/+ and +/+ embryos had closely similar cell proliferation parameters, arguing against a mechanism based on faulty embryonic growth. Thirdly, we tested the hypothesis that the defect in loop-tail results from an inability of the neural folds to become apposed, specifically at the site of Closure 1. By tying a silk suture around the embryonic axis, at the future site of Closure 1, we were able to effect convergence of the neural folds at this site. Neural fold closure failed to progress along the body axis in sutured Lp/Lp embryos, however, in contrast to operated Lp/+ and +/+ embryos which exhibited normal progression of neural tube closure. The embryonic defect in loop-tail appears, therefore, to involve either a general inability of the spinal neural folds to become apposed along the spinal region, or a defect in the process of neural fold fusion.

Clinical trials demonstrate that up to 70% of neural tube defects (NTDs) can be prevented by folic acid supplementation in early pregnancy, whereas the remaining NTDs are resistant to folate. Here, we show that a second vitamin, myo-inositol, is capable of significantly reducing the incidence of spinal NTDs in curly tail mice, a genetic model of folate-resistant NTDs. Inositol increases flux through the inositol/lipid cycle, stimulating protein kinase C activity and upregulating expression of retinoic acid receptor beta, specifically in the caudal portion of the embryonic hindgut. This reduces the delay in closure of the posterior neuropore, the embryonic defect that is known to lead directly to spina bifida in curly tail embryos. Our findings reveal a molecular pathway of NTD prevention and suggest the possible efficacy of combined treatment with folate and inositol in overcoming the majority of human NTDs.

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.

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. Magn Reson Med 73:731–739, 2015. © 2014 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the originalwork is properly cited.

Progression of neurulation in the chick embryo has not been well documented. To provide a detailed description, chick embryos were stained in ovo after the least manipulation possible to avoid distortion of the neural plate and folds. This allowed a morphological and morphometric description of the process of neurulation in relatively undisturbed chick embryos. Neurulation comprises several specific phases with distinct closure patterns and closure rates. The first closure event occurs, de novo, in the future mesencephalon at the 4-6 somite stage (sst 4-6). Soon afterwards, at sst 6-7, de novo closure is seen at the rhombocervical level in the form of multisite contacts of the neural folds. These contacts occur in register with the somites, suggesting that the somites may play a role in forcing elevation and apposition of the neural folds. The mesencephalic] and rhombocervical closure events define an intervening rhombencephalic neuropore, which is present for a brief period before it closes. The remaining pear-shaped posterior neuropore (PNP) narrows and displaces caudally, but its length remains constant in embryos with seven to ten somites, indicating that the caudal extension of the rhombocervical closure point and elongation of the caudal neural plate are keeping pace with each other. From sst 10 onward, the tapered cranial portion of the PNP closes fast in a zipper-like manner, and, subsequently, the wide caudal portion of the PNP closes rapidly as a result of the parallel alignment of its folds, with numerous button-like temporary contact points. A role for convergent extension in this closure event is suggested. The final remnant of the PNP closes at sst 18. Thus, as in mammals, chick neurulation involves multisite closure and probably results form several different development mechanisms at varying levels of the body axis.

The iron-binding growth factor transferrin is taken up and localised in the hindgut of midgestation mouse embryos. We investigated whether the distribution of transferrin may be disturbed in mutant curly tail embryos, a proportion of which exhibit a cell proliferation defect affecting the hindgut endoderm, as part of the pathogenetic sequence leading to development of neural tube defects. Immunostaining revealed a reduction in the binding and/or uptake of transferrin by hindgut epithelial cells in affected curly tail embryos compared with their unaffected littermates. There was no apparent difference between the two embryo types, however, in the distribution or level of expression of the transferrin receptor. The receptor is expressed specifically in the hindgut endoderm of the 10.5-day embryo, although its mRNA is present in all tissues of the posterior neuropore region, suggesting posttranscriptional control of gene expression. These findings may indicate a role for transferrin binding and/or uptake in the regulation of cell proliferation in the hindgut endoderm, with a defect in this process in the curly tail mutant. However, an alternative explanation is suggested by our finding that transferrin immunostaining is more intense in the hindgut of unaffected curly tail embryos than in nonmutant CBA/Ca and CD-1 embryos. Thus, mutant embryos may increase their uptake of transferrin in an attempt to compensate for defective cell proliferation in the hindgut resulting from a defect in another pathway. Only a proportion of embryos are able to mount this compensatory response leading to the observed partial penetrance of developmental defects in the curly tail mutant mouse.

We report seven cases of rare high cervical split spinal cord associated with extensive vertebral fusions (Klippel-Feil anomaly). In light of previous embryological theories and recent research findings we attempt to explain the origin of split cord and vertebral fusions. Two distinctly separate mechanisms are suggested for the development of split cords observed in our cases: a midline lesion bisecting the neuroepithelium and the notochordal plate could be responsible for complete splitting of the cervical cord with anterior bony defect while a localized disturbance of cervical neural tube closure would account for cases with partial dorsal splitting of the cord with posterior vertebral defect. Vertebral fusion anomalies are likely to be associated with disturbance of Pax-1 gene expression in the developing vertebral column. We confirm with our cases the frequent association of failure of normal segmentation and split cord in the cervical region. Clinically, only three patients had neurological deficit which was mild and has remained stable, and they had no radiological evidence of tethering; the minimal disproportionate growth of the cord and spine and the rarity of a bony spur in the cervical region are the likely reasons. A conservative policy was therefore pursued in these cases with careful long-term follow-up.

A genetic basis for neural tube defects (NTD) is rarely doubted, but the genes involved have not yet been identified. This is partly due to a lack of suitable families on which to perform linkage analysis. An alternative approach is to use the many mouse genes that cause NTD as a means of isolating their human homologues. Loop-tail (Lp) is a semidominant mouse gene that, in homozygous mutants, causes the severe NTD phenotype cranio-rachischisis. As a first step toward cloning Lp, we have performed linkage analysis on an intraspecific backcross, using microsatellite and RFLP DNA markers. This study has localized Lp to a region of approximately 1.46 cM on mouse chromosome 1, flanked by the gene for the {alpha} chain of high-affinity Fc receptor for IgE (Fcer1{alpha}) and a microsatellite repeat D1Mit113. Physical mapping data in the region suggest that the interval is likely to be no more than 1.8 Mb in size. The localization is several centimorgans distal to that previously assigned by linkage studies with biochemical and visible markers and suggests that the human homologue of Lp is likely to reside in a region of conserved homology on 1q21-q23. 36 refs., 3 figs., 3 tabs.

A survey of mouse gene knockouts, transgene insertions and spontaneous mutations that are lethal prenatally reveals that surprisingly few developmental disturbances lead to death of the embryo and early foetus. These disturbances include failure to establish and maintain a vascular circulation, and failure to make the transition from yolk-sac-based to liver-based haematopoiesis. The embryo must also establish gestation-dependent routes of nutritional interaction with the mother, including implantation, formation of a yolk-sac vascular circulation, and formation of a chorioallantoic placenta. A number of embryonic organ and body systems, including the central nervous system, gut, lungs, urogenital system and musculoskeletal system, appear to have little or no survival value in utero.

A role for all-trans-retinoic acid in spinal neurulation is suggested by: (1) the reciprocal domains of expression of the retinoic acid receptors RAR-β and RAR-γ in the region of the closed neural tube and open posterior neuropore, respectively, and (2) the preventive effect of maternally administered retinoic acid (5 mg/kg) on spinal neural tube defects in curly tail (ct/ct) mice. Using in situ hybridisation and computerised image analysis we show here that in ct/ct embryos, RAR-β transcripts are deficient in the hindgut endoderm, a tissue whose proliferation rate is abnormal in the ct mutant, and RAR-γ transcripts are deficient in the tail bud and posterior neuropore region. The degree of deficiency of RAR-γ transcripts is correlated with the severity of delay of posterior neuropore closure. As early as 2 hours following RA treatment at 10 days 8 hours post coitum, i.e. well before any morphogenetic effects are detectable, RAR-β expression is specifically upregulated in the hindgut endoderm, and the abnormal expression pattern of RAR-γ is also altered. These results suggest that the spinal neural tube defects which characterise the curly tail phenotype may be due to interaction between the ct gene product and one or more aspects of the retinoic acid signalling pathway.

BACKGROUND: It is widely accepted that periconceptional supplementation with folic acid can prevent a significant proportion of neural tube defects (NTDs). The present study evaluated how folic acid knowledge and periconceptional use for NTD prevention varies by ethnicity in the United Kingdom (U.K.). METHODS: A literature search was conducted to identify studies that included assessment of folic acid knowledge or use in U.K. women of different ethnicities. Only research and referenced sources published after 1991, the year of the landmark Medical Research Council’s Vitamin Study, were included. A meta-analysis was performed of studies that assessed preconceptional folic acid use in Caucasians and non-Caucasians. RESULTS: Five studies met the inclusion criteria for assessment of knowledge and/or use of folic acid supplements in U.K. women including non-Caucasians. The available evidence indicates that South Asians specifically have less knowledge and lower periconceptional use of folic acid than Caucasians; one study found that West Indian and African women also had lower folic acid uptake. A synthesis of results from three of the studies, in a meta-analysis, shows that Caucasians are almost three times more likely to take folic acid before conception than non-Caucasians. CONCLUSION: From the limited evidence available, U.K. women of non-Caucasian ethnicity appear to have less knowledge and a lower uptake of folic acid supplementation than Caucasians during the periconceptional period. Implementing targeted, innovative education campaigns together with a mandatory fortification policy, including the fortification of ethnic minority foods, will be required for maximum prevention of folic acid–preventable NTDs across different ethnic groups. Birth Defects Research (Part A) 97:444–451, 2013. © 2013 Wiley Periodicals, Inc.

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., Author Summary Failure of early development of the central nervous system leads to severe malformations termed neural tube defects (NTDs), including spina bifida and anencephaly. Inherited genetic risk factors play a major role in determining susceptibility to NTDs, but causative genes have proven difficult to identify. In this study we investigated genetic factors that could alter the risk of NTDs in an established mouse model, curly tail, in which defects result from partial loss of function of the grainyhead-like-3 (Grhl3) gene. We identified a variant of lamin B1, a key protein component of the envelope that surrounds the cell nucleus. The protein alteration reduces the structural integrity of the nuclear envelope, causes the nuclei to have altered shape, and reduces the rate of cell division. Curly tail embryos that carry the “abnormal” lamin B1 variant develop NTDs at three times the rate of those that carry the normal version. We conclude that lamin B1 function influences risk of NTDs due to effects on cell proliferation.