Neural tube defects without neural crest defects in splotch mice.

Homozygous Splotch mutant mice (Sp/Sp) die on day 14 of gestation with neural tube defects, curly tail, and malformations of neural crest derivatives. Sp1H mice, which have a radiation-induced allele of Splotch with a similar phenotype, were used for this study. The neural tube defects are always located in the lumbosacral region and in 50% of the cases also in the region of the hindbrain. In this report, rare cases of neural tube defects and tail defects among the offspring of crosses between Splotch (Sp1H) heterozygotes are presented, which are not associated with a neural crest defect. This suggests that the development of the neural tube and neural crest defects in this mutant is caused by independent mechanisms or is dependent on the dosage of the mutant gene, with different thresholds being pathogenetic in the neural tube and neural crest, respectively.     

The Hectd1 ubiquitin ligase is required for development of the head mesenchyme and neural tube closure

Closure of the cranial neural tube depends on normal development of the head mesenchyme. Homozygous-mutant embryos for the ENU-induced open mind (opm) mutation exhibit exencephaly associated with defects in head mesenchyme development and dorsal-lateral hinge point formation. The head mesenchyme in opm mutant embryos is denser than in wildtype embryos and displays an abnormal cellular organization. Since cells that originate from both the cephalic paraxial mesoderm and the neural crest populate the head mesenchyme, we explored the origin of the abnormal head mesenchyme. opm mutant embryos show apparently normal development of neural crest-derived structures. Furthermore, the abnormal head mesenchyme in opm mutant embryos is not derived from the neural crest, but instead expresses molecular markers of cephalic mesoderm. We also report the identification of the opm mutation in the ubiquitously expressed Hectd1 E3 ubiquitin ligase. Two different Hectd1 alleles cause incompletely penetrant neural tube defects in heterozygous animals, indicating that Hectd1 function is required at a critical threshold for neural tube closure. This low penetrance of neural tube defects in embryos heterozygous for Hectd1 mutations suggests that Hectd1 should be considered as candidate susceptibility gene in human neural tube defects.     

jumonji gene is essential for the neurulation and cardiac development of mouse embryos with a C3H/He background.

The recessive mutant mouse jumonji (jmj), obtained by a gene trap strategy, shows neural tube defects in approximately half of homozygous embryos with a BALB/cA and 129/Ola mixed background, but no neural tube defects with BALB/cA, C57BL/6J, and DBA/2J backgrounds. Here, we show that neural tube and cardiac defects are observed in all embryos with a C3H/HeJ background. In addition, abnormal groove formation and prominent flexure are observed on the neural plate with full penetrance, suggesting that abnormal groove formation leads to neural tube defects. We found morphogenetic abnormalities in the bulbus cordis (future outflow tract and the right ventricle) of homozygous embryo hearts. Moreover, myocytes in the ventricular trabeculae show hyperplasia with cells filling the ventricles. Together with the observation that the jmj gene is expressed in the neural epithelium of the head neural plate and in myocytes in the bulbus cordis and trabeculae, the results show that the jmj gene plays essential roles in the normal development of the neural plate, morphogenesis of bulbus cordis, and proliferation of trabecular myocytes on a C3H/He background.     

Dominant Suppressors of Yeast Actin Mutations That Are Reciprocally Suppressed

A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.     

Dorsal patterning defects in the hindbrain, roof plate and skeleton in the dreher (dr(J)) mouse mutant

dreher is a spontaneous mouse mutation in which adult animals display a complex phenotype associated with hearing loss, neurological, pigmentation and skeletal abnormalities. During early embryogenesis, the neural tube of dreher mutants is abnormally shaped in the region of the rhomboencephalon, due to problems in the formation of a proper roof plate over the otic hindbrain. We have studied the expression of Hox/lacZ transgenic mouse strains in the dreher background and shown that primary segmentation of the neural tube is not altered in these mutants, although correct morphogenesis is affected resulting in misshapen rhombomeres. Neural crest derivatives from rhombomere 6, such as the glossopharyngeal ganglion, are defective, and the dorsal neural tube marker Wnt1 is absent from this segment. Selected trunk neural crest populations are also altered, as there is a lack of pigmentation in the thoracic region of mutant mice. Skeletal defects include abnormal cranial bones of neural crest origin, and improper fusion of the dorsal aspects of cervical and thoracic vertebrae. Taken together, the gene affected in the dreher mutant is responsible for correct patterning of the dorsal-most cell types of the neural tube, that is, the neural crest and the roof plate, in the hindbrain region. Axial skeletal defects could reflect inductive influence of the dorsal neural tube on proper fusion of the neural arches. It is possible that a common precursor population for both neural crest and roof plate is the cellular target of the dreher mutation.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.     

Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.     

Neural tube defects in curly-tail mice. I. Incidence, expression and similarity to the human condition.

The incidence of neurovertebral defects in mutant mice of the curly-tail strain was investigated and found to be similar to that observed in the same mice twenty-five years ago. The results of breeding experiments support the hypothesis of Grüneberg that the defects in these mice are probably caused by a recessive gene, the expression of which is markedly affected by the genetic background. Selection against the curly-tail phenotype for six generations did not affect the incidence of abnormalities. A marked excess of females was found among exencephalic mice, as among humans with neural tube defects. Similarly, polyhydramnios, hydrocephaly, high levels of amniotic fluid alphafoetoprotein and distinctive, rapidly adhering cells in the amniotic fluid also occurred in these mice, as in humans. The curly-tail mice thus provide a useful model for the investigation of neural tube defects in man.     

还原叶酸载体基因（RFC1）与神经管和颅面畸形病因学关系的研究进展

神经管畸形和颅面畸形是最常见的出生缺陷,由遗传和环境因素共同作用所致,大规模的人群流行病学研究已证实,叶酸能降低发生这类畸形的危险。叶酸缺乏是神经管和颅面畸形发生的主要环境因素,但其机制尚不清楚,通过对与叶酸代谢有关的还原叶酸载体（reduced folate carrier,RFC）的生化特点、生理功能、还原叶酸载体基因（RFC1）结构功能、调控、表达及其与叶酸水平和神经管颅面畸形的关系等研究进展进行综述,从而为神经管和颅面畸形的病因学研究提出可能的候选基因。 Abstract: Neural tube and craniofacial defects are common birth defects which are ascribed to the combination of genetic and environmental factors. The population epidemiological studies suggested that periconceptional use of multivitamins containing folic acid can reduce a woman’s risk of having a child with neural tube and craniofacial defects. It’s a major environmental factor that periconceptinal women with deficiency of folic acid may increase their risk for delivering babies with neural tube and craniofacial defects, but the mechanism by which folic acid facilitated this risk rediction is unknown. This paper reviews folate transport carrier, Reduced Folate Carrier(RFC)’s characteristics in biological chemistry, physiological function, the folate transport mechanism, structure, function, regulation and expression of reduced folate carrier gene(RFC1), and the relationship between RFC1 with plasm or erythrocyte folate level and neural tube defects, et al. It is suggested a etiologic hypothesis in investigation of candidate gene encoding specific folat-related pathways of neural tube and craniofacial defects.     

Mutations in the Saccharomyces Cerevisiae Type 2a Protein Phosphatase Catalytic Subunit Reveal Roles in Cell Wall Integrity, Actin Cytoskeleton Organization and Mitosis

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37°), cells dependent solely on pph22(ts) alleles for PP2A function displayed a rapid arrest of proliferation. Ts(-) pph22 mutant cells underwent lysis at 37°, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts(-) pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37°. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24° to 37°, Ts(-) pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37°, many Ts(-) pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts(-) pph22 mutant cells also accumulated aberrant microtubule structures at 37°, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts(-) pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.     

The cfy mutation disrupts cell divisions in a stage-dependent manner in zebrafish embryos

The zebrafish curly fry (cfy) mutation leads to embryonic lethality and abnormal cell divisions starting at 12-14 h postfertilization (hpf) during neural tube formation. The mitotic defect is seen in a variety of tissues including the central nervous system (CNS). In homozygous mutant embryos, mitoses are disorganized with an increase in mitotic figures throughout the developing neural tube. One consequence of aberrant mitoses in cfy embryos is an increase in cell death. Despite this, patterning of the early CNS is relatively unperturbed with distribution of the early, primary neurons indistinguishable from that of wild-type embryos. At later stages, however, the number of neurons was dramatically decreased throughout the CNS. The effect on neurons in older cfy embryos but not young ones correlates with the time of birth of neurons: primary neurons are born before the action of the cfy gene and later neurons after. Presumably, death of neuronal progenitors that divide beginning at the neural keel stage or death of their neuronal progeny accounts for the diminution of neurons in older mutant embryos. In addition, oligodendrocytes, which also develop late in the CNS, are greatly reduced in number in cfy embryos due to an apparent decrease in oligodendrocyte precursors. Genetic mosaic analysis demonstrates that the mutant phenotype is cell-autonomous. Furthermore, there are no obvious defects in apical/basal polarity within the neuroepithelium, suggesting that the cfy gene is not critical for epithelial polarity and that polarity defects are unlikely to account for the increased mitotic figures in mutants. These results suggest that the cfy gene regulates mitosis perhaps in a stage-dependent manner in vertebrate embryos.     

Spinal ganglia reduction in the splotch-delayed mouse neural tube defect mutant

Splotch and splotch-delayed mutants have anomalies in certain neural crest cell derivatives as well as neural tube defects. A genetic marker was used to identify mutant, heterozygote, and wild-type embryos within a litter, which enabled us to make intergenotypic comparisons. Histological studies of the lumbosacral region of day 15 and day 16 embryos indicated that the splotch-delayed mutant had similar but less severe defects in spinal ganglion development than those reported for splotch (Auerbach: Journal of Experimental Zoology 127:305-329, 1954). The ganglia were extensively reduced in size, residual, or missing in the splotch-delayed mutant, whereas in the splotch mutant, they were virtually nonexistent. Paired comparison analyses showed that all mutant embryos had a significant reduction in their volume of lumbosacral spinal ganglia when compared to their heterozygous and/or wild-type littermates. Also, some heterozygotes were found to have spinal ganglia volumes that were significantly reduced when compared to wild-type embryos. The volume of spinal ganglia was not related to the severity of the neural tube defect. In fact, three mutant embryos, which did not exhibit a neural tube defect, had spinal ganglia volumes comparable to or less than those mutants with open neural tube lesions or curly tails. This shows that the formation of abnormal neural crest cell derivatives is not a result of the neural tube closure defect. We hypothesize that the two anomalies observed in these mutants have a common etiological basis.     

A unique missense allele of BAF155, a core BAF chromatin remodeling complex protein,causes neural tube closure defects in mice

Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155^msp3, a unique ENU‐induced allele in mice. Homozygous Baf155^mps3 embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA‐Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 483–497, 2014     

Cardiac abnormalities cause early lethality of jumonji mutant mice

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.     

A flower-specific cDNA encoding a novel thionin in tobacco

Summary A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42° C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli sec Y24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.     

The Escherichia coli argU10(Ts) phenotype is caused by a reduction in the cellular level of the argU tRNA for the rare codons AGA and AGG

The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNA(Arg) species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30 degrees C. In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation. An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43 degrees C, and this defect is more serious at the higher temperature. The arginine acceptance is also impaired significantly but to similar extents at the two temperatures. An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30 degrees C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated. Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA. At 43 degrees C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes. We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase.     

Identification and characterization of a novel chemically induced allele at the planar cell polarity gene Vangl2

Planar cell polarity (PCP) signaling controls a number of morphogenetic processes including convergent extension during gastrulation and neural tube formation. Defects in this pathway cause neural tube defects (NTD), the most common malformations of the central nervous system. The Looptail (Lp) mutant mouse was the first mammalian mutant implicating a PCP gene (Vangl2) in the pathogenesis of NTD. We report on a novel chemically induced mutant allele at Vangl2 called Curly Bob that causes a missense mutation p.Ile268Asn (I268N) in the Vangl2 protein. This mutant segregates in a semi-dominant fashion with heterozygote mice displaying a looped tail appearance, bobbing head, and a circling behavior. Homozygote mutant embryos suffer from a severe form of NTD called craniorachischisis, severe PCP defects in the inner hair cells of the cochlea and posterior cristae, and display a distinct defect in retinal axon guidance. This mutant genetically interacts with the Lp allele (Vangl2 ^S464N) in neural tube development and inner ear hair cell polarity. The Vangl2^I268N protein variant is expressed at very low levels in affected neural and retinal tissues of mutant homozygote embryos. Biochemical studies show that Vangl2^I268N exhibits impaired targeting to the plasma membrane and accumulates in the endoplasmic reticulum. The Vangl2^I268N variant no longer physically interacts with its PCP partner DVL3 and has a reduced protein half-life. This mutant provides an important model for dissecting the role of Vangl2 in the development of the neural tube, establishment of polarity of sensory cells of the auditory and vestibular systems, and retinal axon guidance.

     

Identification of the mouse Loop‐tail gene: a model for human craniorachischisis?

Neural tube defects are one of the commonest human birth defects, with more than 0.5% of some populations affected. Mouse models are being used in an attempt to identify genes that could be involved in these malformations. Only two mouse mutations are known to lead to craniorachischisis, failure of closure of almost the entire neural tube. Two recent papers report that the gene for one of these, Loop-tail, has now been identified and sequenced.1, 2 It has been given the designation Ltap/Lpp1 and appears to function in floor plate formation. It will be of great interest to investigate the role of this gene in human neural tube defects.