Anticonvulsant profile and teratogenic evaluation of potent new analogues of a valproic acid urea derivative in NMRI mice

BACKGROUND: Valproic acid (VPA) is used to treat epilepsy and bipolar disorders, as well as for migraine prophylaxis. However, its clinical use is limited by two life-threatening side effects: hepatotoxicity and teratogenicity. To develop a more potent and safer second-generation VPA drug, the urea derivatives of four VPA analogs (2-ethyl-3-methylpentanoyl urea, 2-ethylhexanoyl urea, 2-ethyl-4-methylpentanoyl urea, and 2-methylbutanoyl urea) were synthesized. METHODS: Four CNS-active analogs of a VPA urea derivative testedthe anticonvulsant activity in the maximal electroshock seizure test (MES) and subcutaneous metrazol seizure threshold test (scMet). Teratogenic effects of these compounds were evaluated in NMRI mice susceptible to VPA-induced teratogenicity by comparison with VPA. RESULTS: All four VPA analogs showed superior anticonvulsant activity over VPA. Compared with VPA, which induced neural tube defects (NTDs) in fetuses at 1.8 and 3.6 mmol/kg, the analog derivatives induced no NTDs at any concentration up to 4.8 mmol/kg (except for a single abnormality at 3.6 mmol/kg with 2-ethyl-3-methylpentanoyl urea). Skeletal examination also revealed that the acylurea derivatives induced vertebral and rib abnormalities in fetuses markedly less frequently than VPA. Our results confirmed that the analogue derivatives are significantly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS-active VPA analogs containing a urea moiety, which have better anticonvulsant potency and lack teratogenicity, are good potential candidates as second-generation VPA antiepileptic drugs. Birth Defects Res (Part B) 86:394–401, 2009. © 2009 Wiley-Liss, Inc.     

Ribonucleotide reductase subunit R1: a gene conferring sensitivity to valproic acid-induced neural tube defects in mice

Neural tube defects (NTDs), although prevalent and easily diagnosed, are etiologically heterogeneous, rendering mechanistic interpretation problematic. To date, there is evidence that mammalian neural tube closure (NTC) initiates and fuses intermittently at four discrete locations. Disruption of this process at any of these four sites may lead to a region-specific NTDs, possibly arising through closure site-specific genetic mechanisms. Although recent efforts have focused on elucidating the genetic components of NTDs, a void persists regarding gene identification in closure site-specific neural tissue. To this end, experiments were conducted to identify neural tube closure site-specific genes that might confer regional sensitivity to teratogen-induced NTDs. Using an inbred mouse strain (SWV/Fnn) with a high susceptibility to VPA- induced NTDs that specifically targets and disrupts NTC between the prosencephalon and mesencephalon region (future fore/midbrain; neural tube closure site II), we identified a VPA-sensitive closure site II-specific clone. Sequencing of this clone from an SWV neural tube cDNA library confirmed that it encodes the r1 subunit of the cell cycle enzyme ribonucleotide reductase (RNR). The abundance of rnr-r1 mRNA was significantly increased in response to VPA drug treatment. This upregulated expression was accompanied by a significant decrease in cellular proliferation in the closure site II neural tube region of the embryos, as determined by ELISA cellular proliferation assays performed on BrdU-pulsed neuroepithelial cells in vivo. We hypothesize that rnr-r1 plays a critical role in the development of VPA-induced exencephaly.     

Myo-inositol enhances teratogenicity of valproic acid in the mouse

BACKGROUND: Valproic acid (VPA) is an anticonvulsant drug that is widely used therapeutically for a variety of neurological conditions. VPA is also well known for its teratogenic potential in both humans and experimental animal models. The typical malformations observed following VPA exposure include neural tube defects (NTDs) and craniofacial and skeletal malformations. Nevertheless, the mechanisms underlying VPA's anticonvulsant efficacy or its teratogenicity remain to be elucidated. It was recently suggested that a relationship exists between VPA exposure and the cellular depletion of myo-inositol (INO). Furthermore, INO has been shown to rescue NTDs in the curly tail mouse. The aim of this study was to investigate the interactions of VPA and INO in the developing embryo. METHODS: For this purpose, 2 strains of mice were used: SWV/Fnn (known to be sensitive to VPA) and LM/Bc (known to be resistant to VPA-induced NTDs). Pregnant females were randomly assigned to 4 experimental groups: control, VPA (600 mg/kg), INO (400 mg/kg), and VPA plus INO. VPA was injected IP at 8.5 days postcoitum (dpc). INO was administered PO twice a day from 6.5 to 10.5 dpc. At term the dams were killed, the uteri were removed, and all of the general toxicological parameters (number of implants, resorptions, dam weight, and fetus weight) were recorded and statistically analyzed. RESULTS: Postimplantation loss in the SWV/Fnn strain and NTDs in the LM/Bc strain were significantly increased after the coadministration of VPA and INO. CONCLUSIONS: This work clearly indicates that INO enhances VPA-induced teratogenicity in the mouse.     

Ascorbic Acid Reverses Valproic Acid-Induced Inhibition of <Emphasis Type="Italic">Hoxa2</Emphasis> and Maintains Glutathione Homeostasis in Mouse Embryos in Culture

Valproic acid (VPA) has been shown to cause neural tube defects in humans and mice, but its mechanism of action has not been elucidated. We hypothesize that alterations in embryonic antioxidant status and Hoxa2 gene expression play an important role in VPA-induced teratogenesis. A whole embryo culture system was applied to explore the effects of VPA on total glutathione, on glutathione in its oxidized (GSSG) and reduced (GSH) forms [GSSG/GSH ratio] and on Hoxa2 expression in cultured CD-1 mouse embryos during their critical period of organogenesis. Our results show that VPA can (1) induce embryo malformations including neural tube defects, abnormal flexion, yolk sac circulation defects, somite defects, and craniofacial deformities such as fusion of the first and second arches, and (2) alter glutathione homeostasis of embryos through an increase in embryonic GSSG/GSH ratio and a decrease in total GSH content in embryos. Western blot analysis and quantitative real-time RT-PCR show that VPA can inhibit Hoxa2 expression in cultured embryos at both the protein and mRNA level, respectively. The presence of ascorbic acid in the culture media was effective in protecting embryos against oxidative stress induced by VPA and prevented VPA-induced inhibition of Hoxa2 gene expression. Hoxa2 null mutant embryos do not exhibit altered glutathione homeostasis, indicating that inhibition of Hoxa2 is downstream of VPA-induced oxidative stress. These results are first to suggest VPA may, in part, exert its teratogenicity through alteration of the embryonic antioxidant status and inhibition of Hoxa2 gene expression and that ascorbic acid can protect embryos from VPA-induced oxidative stress.     

Amidic modification of valproic acid reduces skeletal teratogenicity in mice

BACKGROUND: The antiepileptic drug valproic acid (VPA) is well known to cause neural tube and skeletal defects in both humans and animals. The amidic VPA analogues valpromide (VPD) and valnoctamide (VCD) have much lower teratogenicity than VPA inducing exencephaly in mice. The objective of this study was to investigate the teratogenic effects of VPA, VPD, and VCD on the skeleton of NMRI mice. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of VPA (400 and 800 mg/kg), VPD (800 mg/kg), or VCD (800 mg/kg) on the morning of gestation day (GD) 8. Cesarean section was carried out on GD 18. Live fetuses were double‐stained for bone and cartilage and their skeletons were examined. RESULTS: Significant increases in fetal loss and exencephaly rate were observed with VPA at 800 mg/kg compared to the vehicle control. There were no significant differences between either VPD or VCD and the control groups for any parameter at cesarean section. A number of abnormalities were dose‐dependently induced at high incidences by VPA in both the cartilage and bone of vertebrae, ribs and sternum. In contrast, lower frequencies of abnormality were exhibited with VPD and VCD than VPA in all skeletons affected by VPA. CONCLUSIONS: These findings clearly indicate that VPD and VCD are distinctly less teratogenic than VPA in the induction of not only neural tube defects, but also skeletal abnormalities. A structure‐teratogenicity relationship of VPA on the skeleton is suspected. Birth Defects Res B 71:47–53, 2004. © 2004 Wiley‐Liss, Inc.     

Mini-review: toward understanding mechanisms of genetic neural tube defects in mice.

We review the data from studies of mouse mutants that lend insight to the mechanisms that lead to neural tube defects (NTDs). Most of the 50 single-gene mutations that cause neural tube defects (NTDs) in mice also cause severe embryonic-lethal syndromes, in which exencephaly is a nonspecific feature. In a few mutants (e.g., Trp53, Macs, Mlp or Sp), other defects may be present, but affected fetuses can survive to birth. Multifactorial genetic causes, as are present in the curly tail stock (15-20% spina bifida), or the SELH/Bc strain (15-20% exencephaly), lead to nonsyndromic NTDs. The mutations indicate that "spina bifida occulta," a dorsal gap in the vertebral arches over an intact neural tube, is usually genetically and developmentally unrelated to exencephaly or "spina bifida" (aperta). Almost all exencephaly or spina bifida aperta of genetic origin is caused by failure of neural fold elevation. The developmental mechanisms in genetic NTDs are considered in terms of distinct rostro-caudal zones along the neural folds that likely differ in mechanism of elevation. Failure of elevation leads to: split face (zone A), exencephaly (zone B), rachischisis (all of zone D), or spina bifida (caudal zone D). The developmental mechanisms leading to these genetic NTDs are heterogeneous, even within one zone. At the tissue level, the mutants show that the mechanism of failure of elevation can involve, e.g., (1) slow growth of adjacent tethered tissue (curly tail), (2) defective forebrain mesenchyme (Cart1 or twist), (3) defective basal lamina in surface ectoderm (Lama5), (4) excessive breadth of floorplate and notochord (Lp), (5) abnormal neuroepithelium (Apob, Sp, Tcfap2a), (6) morphological deformation of neural folds (jmj), (7) abnormal neuroepithelial and neural crest cell gap-junction communication (Gja1), or (8) incomplete compensation for a defective step in the elevation sequence (SELH/Bc). At the biochemical level, mutants suggest involvement of: (1) faulty regulation of apoptosis (Trp53 or p300), (2) premature differentiation (Hes1), (3) disruption of actin function (Macs or Mlp), (4) abnormal telomerase complex (Terc), or (5) faulty pyrimidine synthesis (Sp). The NTD preventative effect of maternal dietary supplementation is also heterogeneous, as demonstrated by: (1) methionine (Axd), (2) folic acid or thymidine (Sp), or (3) inositol (curly tail). The heterogeneity of mechanism of mouse NTDs suggests that human NTDs, including the common nonsyndromic anencephaly or spina bifida, may also reflect a variety of genetically caused defects in developmental mechanisms normally responsible for elevation of the neural folds.     

Tetramethylcyclopropyl analogue of the leading antiepileptic drug, valproic acid: evaluation of the teratogenic effects of its amide derivatives in NMRI mice

BACKGROUND: Although valproic acid (VPA) is used extensively for treating various kinds of epilepsy, it causes hepatotoxicity and teratogenicity. In an attempt to develop a more potent and safer second generation to VPA drug, the amide derivatives of the tetramethylcyclopropyl VPA analogue, 2,2,3,3‐tetramethylcyclopropanecarboxamide (TMCD), N‐methyl‐TMCD (MTMCD), 4‐(2,2,3,3‐tetramethylcyclopropanecarboxamide)‐benzenesulfonamide (TMCD‐benzenesulfonamide), and 5‐(TMCD)‐1,3,4‐thiadiazole‐2‐sulfonamide (TMCD‐thiadiazolesulfonamide) were synthesized and shown to have more potent anticonvulsant activity than VPA. Teratogenic effects of these CNS‐active compounds were evaluated in Naval Medical Research Institute (NMRI) mice susceptible to VPA‐induced teratogenicity by comparing them to those of VPA. METHODS: Pregnant NMRI mice were given a single sc injection of either VPA or TMC‐amide derivatives on gestation day 8.5, and then the live fetuses were examined to detect any external malformations on gestation day 18. After double‐staining for bone and cartilage, their skeletons were examined. RESULTS: In contrast to VPA, which induced NTDs in a high number of fetuses at 2.4–4.8 mmol/kg, TMCD, TMCD‐benzenesulfonamide, and TMCD‐thiadiazolesulfonamide at 4.8 mmol/kg and MTMCD at 3.6 mmol/kg did not induce a significant number of NTDs. TMCD‐thiadiazolesulfonamide exhibited a potential to induce limb defects in fetuses. Skeletal examination also revealed that fetuses exposed to all four of the tetramethylcyclopropanecarboxamide derivatives developed vertebral and rib abnormalities less frequently than those exposed to VPA. Our results established that TMCD, MTMCD, and TMCD‐benzenesulfonamide are distinctly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS‐active amides containing a tetramethylcyclopropanecarbonyl moiety demonstrated better anticonvulsant potency compared to VPA and a lack of teratogenicity, which makes these compounds good second‐generation VPA antiepileptic drug candidates. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Valproic acid-induced neural tube defects: reduction by folinic acid in the mouse

Neural tube defects were induced dose-dependently by single injections of the anticonvulsant drug valproic acid (VPA) as sodium salt in mice on gestational day 8. Folinic acid (5-CHO-THF) coadministration by i.p. injection or by a constant rate infusion via osmotic minipumps, implanted s.c., significantly reduced the exencephaly rates using a randomized double-blind experimental procedure. 5-CHO-THF supplementation cut the exencephaly rates into half even at high maternal plasma levels of VPA (p less than 0.005, chi 2-test); resorption rates were not affected. The VPA plasma kinetics were not changed by any of the application regimens of 5-CHO-THF. The investigation of the folate metabolite pattern (determined by HPLC) showed that 5-CHO-THF and 5-methyl-tetrahydrofolic acid (5-CH3-THF) were the main metabolites in untreated mice. After supplementation with 5-CHO-THF, only the concentrations of this folate vitamer were increased in the plasma from 0.3 microgram/ml (normal) to 0.6 or 1.9 micrograms/ml (after injection of 3 x 1 mg/kg or 3 X 4 mg/kg) and to 4.2 micrograms/ml (after infusion via osmotic minipumps). Our results indicate that VPA-induced exencephaly in mice combined with the investigation of the plasma levels of VPA and the different folate metabolites could be an appropriate animal model to study protective effects of folates on the occurrence of neural tube defects.     

Genetic analysis of the cause of exencephaly in the SELH/Bc mouse stock

A new mouse stock, SELH/Bc, having a high liability to exencephaly has been developed. About 17% of SELH fetuses are exencephalic. The genetic cause of this exencephaly was investigated in a cross to a normal related ICR/Bc strain and in subsequent classical genetic crosses (F2, first and second backcrosses). The data were compared with a number of genetic models, including that of a single recessive mutation with 17% penetrance. The data did not fit single-locus inheritance. The expectations from the multifactorial threshold model based on an underlying quantitative liability trait with additive inheritance were found to fit the data very well. The number of loci involved was estimated to be about two or three. About 70% of exencephalic SELH fetuses are female, and there is no overall deficiency of males. The relatively higher risk in females was constant across the genetic backgrounds in the experiment. In summary, the liability to exencephaly in SELH mice appears to be a multifactorial threshold trait, and it therefore resembles human neural tube defects in type of genetic etiology. SELH therefore may be a valuable animal model in the study of neural tube defects.     

Valproic acid increases expression of methylenetetrahydrofolate reductase (MTHFR) and induces lower teratogenicity in MTHFR deficiency

Valproate (VPA) treatment in pregnancy leads to congenital anomalies, possibly by disrupting folate or homocysteine metabolism. Since methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of folate interconversion and homocysteine metabolism, we addressed the possibility that VPA might have different teratogenicity in Mthfr(+/+) and Mthfr(+/-) mice and that VPA might interfere with folate metabolism through MTHFR modulation. Mthfr(+/+) and Mthfr(+/-) pregnant mice were injected with VPA on gestational day 8.5; resorption rates and occurrence of neural tube defects (NTDs) were examined on gestational day 14.5. We also examined the effects of VPA on MTHFR expression in HepG2 cells and on MTHFR activity and homocysteine levels in mice. Mthfr(+/+) mice had increased resorption rates (36%) after VPA treatment, compared to saline treatment (10%), whereas resorption rates were similar in Mthfr(+/-) mice with the two treatments (25-27%). NTDs were only observed in one group (VPA-treated Mthfr(+/+)). In HepG2 cells, VPA increased MTHFR promoter activity and MTHFR mRNA and protein (2.5- and 3.7-fold, respectively). Consistent with cellular MTHFR upregulation by VPA, brain MTHFR enzyme activity was increased and plasma homocysteine was decreased in VPA-treated pregnant mice compared to saline-treated animals. These results underscore the importance of folate interconversion in VPA-induced teratogenicity, since VPA increases MTHFR expression and has lower teratogenic potential in MTHFR deficiency.     

Aberrant induction of p19Arf-mediated cellular senescence contributes to neurodevelopmental defects

Valproic acid (VPA) is a widely prescribed drug to treat epilepsy, bipolar disorder, and migraine. If taken during pregnancy, however, exposure to the developing embryo can cause birth defects, cognitive impairment, and autism spectrum disorder. How VPA causes these developmental defects remains unknown. We used embryonic mice and human organoids to model key features of VPA drug exposure, including exencephaly, microcephaly, and spinal defects. In the malformed tissues, in which neurogenesis is defective, we find pronounced induction of cellular senescence in the neuroepithelial (NE) cells. Critically, through genetic and functional studies, we identified p19^Arf as the instrumental mediator of senescence and microcephaly, but, surprisingly, not exencephaly and spinal defects. Together, these findings demonstrate that misregulated senescence in NE cells can contribute to developmental defects.

Does cellular senescence contribute to developmental birth defects? This study shows that valproic acid induces cellular senescence in neuroepithelial cells, the precursors of all adult brain cells, and blocks their differentiation, suggesting that aberrant senescence in the embryo may contribute to postnatal cognitive defects.     

Orally Administered Melatonin Prevents Lipopolysaccharide-Induced Neural Tube Defects in Mice

Lipopolysaccharide (LPS) has been associated with adverse pregnant outcomes, including fetal demise, intra-uterine growth restriction (IUGR), neural tube defects (NTDs) and preterm delivery in rodent animals. Previous studies demonstrated that melatonin protected against LPS-induced fetal demise, IUGR and preterm delivery. The aim of the present study was to investigate the effects of melatonin on LPS-induced NTDs. All pregnant mice except controls were intraperitoneally injected with LPS (25 µg/kg) daily from gestational day (GD)8 to GD12. Some pregnant mice were orally administered with melatonin (MT, 50 mg/kg) before each LPS injection. A five-day LPS injection resulted in 27.5% of fetuses with anencephaly, exencephaly or encephalomeningocele. Additional experiment showed that maternal LPS exposure significantly down-regulated placental proton-coupled folate transporter (pcft) and disturbed folate transport from maternal circulation through the placentas into the fetus. Interestingly, melatonin significantly attenuated LPS-induced down-regulation of placental pcft. Moreover, melatonin markedly improved the transport of folate from maternal circulation through the placentas into the fetus. Correspondingly, orally administered melatonin reduced the incidence of LPS-induced anencephaly, exencephaly or encephalomeningocele. Taken together, these results suggest that orally administered melatonin prevents LPS-induced NTDs through alleviating LPS-induced disturbance of folate transport from maternal circulation through the placenta into the fetus.     

Genetic identification of a novel bolting locus in <Emphasis Type="Italic">Beta vulgaris</Emphasis> which promotes annuality independently of the bolting gene <Emphasis Type="Italic">B</Emphasis>

Bolting tendency in the crop species Beta vulgaris, which includes sugar beet, is a complex trait governed by various environmental cues, including prolonged periods of cold temperatures over winter (vernalization) and photoperiod, and multiple genetic factors. Two loci which promote bolting in the absence of vernalization are known in beet, the major bolting locus B on chromosome II and the B2 locus on chromosome IX. Here, genetic linkage and quantitative trait locus analyses in two populations derived from a cross between a biennial genotype, which was identified in a phenotypic screen for EMS-induced bolting mutants and requires vernalization to bolt, and an annual wild beet accession revealed the presence of a novel major bolting locus B4 which is linked to the B locus but promotes annual bolting independently of B. The genetic distance between B and B4 on chromosome II is 11 cM. A sequence-based marker was identified which co-segregates with bolting behavior and co-localizes with the B4 locus.     

Global DNA hypomethylation is associated with NTD‐affected pregnancy: A case‐control study

BACKGROUND: Neural tube defects are severe, common birth defects that result from failure of neural tube closure. They are considered to be a multifactorial disorder, and our knowledge of causal mechanisms remains limited. We hypothesized that abnormal DNA methylation occurs in NTD‐affected fetuses. The correlations of global DNA methylation levels with complexity of NTDs and known risk factors of NTDs, MTHFR genotype and fever, were analyzed. METHODS: A hospital‐based case‐control study was performed. Epidemiologic data, pathologic diagnosis, and methylenetetrahydrofolate reductase (MTHFR) genotype analysis were completed. Array comparative genomic hybridization was used to exclude cytogenetic abnormalities. Global DNA methylation statuses were determined for both brain and skin tissue. RESULTS: Sixty‐five NTD‐affected fetuses and 65 normal controls matched for gestational and maternal ages were collected. In brain tissue, global DNA methylation levels were significantly decreased in cases compared with controls (4.12 vs. 4.99%; p < 0.001). DNA hypomethylation (<4.35%) resulted in a significant 5.736‐fold increased risk for NTDs (95% confidence interval, 1.731–19.009; p = 0.004). Nonisolated NTDs had lower levels of global DNA methylation than did isolated NTDs (3.77 vs. 4.70%; p = 0.022). After stratifying subjects by MTHFR genotype, we observed a skewed distribution of global DNA methylation levels. For genotype C/C, global DNA methylation status was the same in the two groups (4.51 vs. 4.72%; p = 0.687). For T/T, cases had significantly lower global methylation levels than did controls (5.23 vs. 3.79%; p < 0.001). CONCLUSIONS: Global DNA hypomethylation in fetal brain tissue was associated with NTD‐affected pregnancy. DNA methylation levels were correlated with NTD complexity. The MTHFR genotype contributed to global DNA hypomethylation. Birth Defects Research (Part A), 2010. © 2010 Wiley‐Liss, Inc.     

Auxotrophic basis for the envelope-defective phenotype of anEscherichia coli mutant

A mutant ofEscherichia coli K-12, originally though to lack the major murein lipoprotein (product of the1pp gene) was found to contain an intact1pp locus and to exhibit multiple physiological defects. These included altered morphology, sensitivity to glycine, sensitivity to high temperature, and absolute requirement for certain vitamin B₆ derivatives. The genetic properties of the mutant indicated that a chromosomal inversion had caused inactivation of thepdxH (pyridoxine phosphate oxidase) gene. Behavior of this and other Pdx^– mutants indicated that growth in unsupplemented complex medium can lead to pyridoxal phosphate limitation and concomitant impairment of cell wall biosynthesis.     

Genetic Mapping and QTL Analysis of Growth-Related Traits in the Pacific Oyster

The Pacific oyster (Crassostrea gigas) is one of the most important oysters cultured worldwide. To analyze the oyster genome and dissect growth-related traits, we constructed a sex-averaged linkage map by combining 64 genomic simple sequence repeats, 42 expressed sequence tag-derived SSRs, and 320 amplified fragment length polymorphism markers in an F₁ full-sib family. A total of 426 markers were assigned to 11 linkage groups, spanning 558.2 cM with an average interval of 1.3 cM and 94.7% of genome coverage. Segregation distortion was significant for 18.8% of the markers (P < 0.05), and distorted markers tended to occur on some genetic regions or linkage groups. Most growth-related quantitative traits were highly significantly (P < 0.01) correlated, and principal component analysis obtained four principal components. Quantitative trait locus (QTL) analysis identified three significant QTLs for two principal components, which explained 0.6–13.9% of the phenotypic variation. One QTL for sex was detected on linkage group 6, and the inheritabilities of sex for parental alleles and maternal alleles on that locus C15 are 39.8% and 0.01%, respectively. The constructed linkage map and determined QTLs can provide a tool for further genetic analysis of the traits and be potential for marker-assisted selection in C. gigas breeding.     

A first set of gene-derived polymorphic microsatellite markers in half-smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Half-smooth tongue sole (Cynoglossus semilaevis) is a commercially important marine fish species in China. A set of type I microsatellite markers were identified through bioinformatic mining of the GenBank database. Thirteen of these markers showed polymorphisms through genotyping a sample of 30 individuals. A total of 47 alleles were detected, with an average of 3.6 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to five, from 0.14 to 0.93 and from 0.27 to 0.77, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0038) and no significant linkage disequilibrum between pairs of loci was found. The markers identified in this study will contribute to construction of genetic linkage maps and mapping of quantitative trait loci (QTL) of C. semilaevis.     

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     

Genetic transactivation of Dissociation elements in transgenic tomato plants

Summary A DNA amplification is correlated with the dominant, unstable cob-354 cobalt resistance trait in the cellular slime mold, Dictyostelium discoideum. The amplified DNA is present as about 50 copies of an extrachromosomal element. Cells grown under nonselective conditions in the absence of cobalt ions lose both the cobalt resistance trait and all extrachromosomal copies of the amplified DNA. The amplified DNA is transferrable to new genetic backgrounds by parasexual genetic crosses. These results explain the inability to map the cob-354 trait to a linkage group. The chromosomal origin of the amplified DNA is group III or VI. Thus the resistance trait appears to be independent of the previously known cobalt resistance locus, cobA, which maps to group VII. A developmental defect involving the production of multiply-tipped aggregates that do not complete fruiting body formation also is correlated with the presence of the amplified DNA.