Cellular mechanisms underlying Pax3-related neural tube defects and their prevention by folic acid.

Neural tube defects (NTDs), including spina bifida and anencephaly, are among the most common birth defects worldwide, but their underlying genetic and cellular causes are not well understood. Some NTDs are preventable by supplemental folic acid. However, despite widespread use of folic acid supplements and implementation of food fortification in many countries, the protective mechanism is unclear. Pax3 mutant (splotch; Sp2H ) mice provide a model in which NTDs are preventable by folic acid and exacerbated by maternal folate deficiency. Here, we found that cell proliferation was diminished in the dorsal neuroepithelium of mutant embryos, corresponding to the region of abolished Pax3 function. This was accompanied by premature neuronal differentiation in the prospective midbrain. Contrary to previous reports, we did not find evidence that increased apoptosis could underlie failed neural tube closure in Pax3 mutant embryos, nor that inhibition of apoptosis could prevent NTDs. These findings suggest that Pax3 functions to maintain the neuroepithelium in a proliferative, undifferentiated state, allowing neurulation to proceed. NTDs in Pax3 mutants were not associated with abnormal abundance of specific folates and were not prevented by formate, a one-carbon donor to folate metabolism. Supplemental folic acid restored proliferation in the cranial neuroepithelium. This effect was mediated by enhanced progression of the cell cycle from S to G2 phase, specifically in the Pax3 mutant dorsal neuroepithelium. We propose that the cell-cycle-promoting effect of folic acid compensates for the loss of Pax3 and thereby prevents cranial NTDs.

Aberrant methylation of Pax3 gene and neural tube defects in association with exposure to polycyclic aromatic hydrocarbons.

BACKGROUND: Neural tube defects (NTDs) are common and severe congenital malformations. Pax3 is an essential gene for neural tube closure in mice but it is unknown whether altered expression or methylation of PAX3 contributes to human NTDs. We examined the potential role of hypermethylation of Pax3 in the development of NTDs by analyzing human NTD cases and a mouse model in which NTDs were induced by benzo[a]pyrene (BaP), a widely studied polycyclic aromatic hydrocarbon (PAH). METHODS: We extracted methylation information of PAX3 in neural tissues from array data of ten NTD cases and eight non-malformed controls. A validation study was then performed in a larger independent population comprising 73 NTD cases and 29 controls. Finally, we examined methylation patterns and expression of Pax3 in neural tissues from mouse embryos of dams exposed to BaP or BaP and vitamin E. RESULTS: Seven CpG sites in PAX3 were hypermethylated in NTD fetuses as compared to controls in the array data. In the validation phase, significantly higher methylation levels in the body region of PAX3 were observed in NTD cases than in controls (P = 0.003). And mean methylation intensity in the body region of PAX3 in fetal neural tissues was positively correlated with median concentrations of PAH in maternal serum. In the mouse model, BaP-induced NTDs were associated with hypermethylation of specific CpG sites within both the promoter and body region of Pax3. Supplementation with vitamin E via chow decreased the rate of NTDs, partly recovered the repressed total antioxidant capacity in mouse embryos exposed to BaP, and this was accompanied by the normalization of Pax3 methylation level and gene expression. CONCLUSION: Hypermethylation of Pax3 may play a role in the development of NTDs; DNA methylation aberration may be caused by exposure to BaP, with possible involvement of oxidative stress.

Downregulation of α-Melanocyte-Stimulating Hormone-Induced Activation of the Pax3-MITF-Tyrosinase Axis by Sorghum Ethanolic Extract in B16F10 Melanoma Cells.

Ultraviolet irradiation-induced hyperpigmentation of the skin is associated with excessive melanin production in melanocytes. Tyrosinase (TYR) is a key enzyme catalyzing the rate-limiting step in melanogenesis. TYR expression is controlled by microphthalmia-associated transcription factor (MITF) expression. Sorghum is a cereal crop widely used in a variety of foods worldwide. Sorghum contains many bioactive compounds and is beneficial to human health. However, the effects of sorghum in anti-melanogenesis have not been well characterized. In this study, the biological activity of sorghum ethanolic extract (SEE) on α-melanocyte-stimulating hormone (α-MSH)-induced TYR expression was evaluated in B16F10 melanoma cells. SEE attenuated α-MSH-induced TYR gene promoter activity through the downregulation of the transcription factor MITF. We found that paired box gene 3 (Pax3) contributes to the maximal induction of MITF gene promoter activity. Further analysis demonstrated that SEE inhibited α-MSH-induced Pax3 expression. The collective results indicate that SEE attenuates α-MSH-induced TYR expression through the suppression of Pax3-mediated MITF gene promoter activity. Targeting the Pax3-MITF axis pathway could be considered a potential strategy to increase the efficacy of anti-melanogenesis.

Folic Acid Exposure Rescues Spina Bifida Aperta Phenotypes in Human Induced Pluripotent Stem Cell Model.

Neural tube defects (NTDs) are severe congenital abnormalities, caused by failed closure of neural tube during early embryonic development. Periconceptional folic acid (FA) supplementation greatly reduces the risk of NTDs. However, the molecular mechanisms behind NTDs and the preventive role of FA remain unclear. Here, we use human induced pluripotent stem cells (iPSCs) derived from fetuses with spina bifida aperta (SBA) to study the pathophysiology of NTDs and explore the effects of FA exposure. We report that FA exposure in SBA model is necessary for the proper formation and maturation of neural tube structures and robust differentiation of mesodermal derivatives. Additionally, we show that the folate antagonist methotrexate dramatically affects the formation of neural tube structures and FA partially reverts this aberrant phenotype. In conclusion, we present a novel model for human NTDs and provide evidence that it is a powerful tool to investigate the molecular mechanisms underlying NTDs, test drugs for therapeutic approaches.

Folate Receptor Alpha Upregulates Oct4, Sox2 and Klf4 and Downregulates miR-138 and miR-let-7 in Cranial Neural Crest Cells.

Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5' enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp-/- ), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016;34:2721-2732.

The green tea polyphenol EGCG alleviates maternal diabetes-induced neural tube defects by inhibiting DNA hypermethylation.

BACKGROUND: Maternal diabetes increases the risk of neural tube defects in offspring. Our previous study demonstrated that the green tea polyphenol, Epigallocatechin gallate, inhibits high glucose-induced neural tube defects in cultured embryos. However, the therapeutic effect of Epigallocatechin gallate on maternal diabetes-induced neural tube defects is still unclear. OBJECTIVE: We aimed to examine whether Epigallocatechin gallate treatment can reduce maternal diabetes-induced DNA methylation and neural tube defects. STUDY DESIGN: Nondiabetic and diabetic pregnant mice at embryonic day 5.5 were given drinking water with or without 1 or 10 µM Epigallocatechin gallate. At embryonic day 8.75, embryos were dissected from the visceral yolk sac for the measurement of the levels and activity of DNA methyltransferases, the levels of global DNA methylation, and methylation in the CpG islands of neural tube closure essential gene promoters. embryonic day 10.5 embryos were examined for neural tube defect incidence. RESULTS: Epigallocatechin gallate treatment did not affect embryonic development because embryos from nondiabetic dams treated with Epigallocatechin gallate did not exhibit any neural tube defects. Treatment with 1 µM Epigallocatechin gallate did not reduce maternal diabetes-induced neural tube defects significantly. Embryos from diabetic dams treated with 10 µM Epigallocatechin gallate had a significantly lower neural tube defect incidence compared with that of embryos without Epigallocatechin gallate treatment. Epigallocatechin gallate reduced neural tube defect rates from 29.5% to 2%, an incidence that is comparable with that of embryos from nondiabetic dams. Ten micromoles of Epigallocatechin gallate treatment blocked maternal diabetes-increased DNA methyltransferases 3a and 3b expression and their activities, leading to the suppression of global DNA hypermethylation. Additionally, 10 µM Epigallocatechin gallate abrogated maternal diabetes-increased DNA methylation in the CpG islands of neural tube closure essential genes, including Grhl3, Pax3, and Tulp3. CONCLUSION: Epigallocatechin gallate reduces maternal diabetes-induced neural tube defects formation and blocks the enhanced expression and activity of DNA methyltransferases, leading to the suppression of DNA hypermethylation and the restoration of neural tube closure essential gene expression. These observations suggest that Epigallocatechin gallate supplements could mitigate the teratogenic effects of hyperglycemia on the developing embryo and prevent diabetes-induced neural tube defects.

Pax3 synergizes with Gli2 and Zic1 in transactivating the Myf5 epaxial somite enhancer.

Both Glis, the downstream effectors of hedgehog signaling, and Zic transcription factors are required for Myf5 expression in the epaxial somite. Here we demonstrate a novel synergistic interaction between members of both families and Pax3, a paired-domain transcription factor that is essential for both myogenesis and neural crest development. We show that Pax3 synergizes with both Gli2 and Zic1 in transactivating the Myf5 epaxial somite (ES) enhancer in concert with the Myf5 promoter. This synergy is dependent on conserved functional domains of the proteins, as well as on a novel homeodomain motif in the Myf5 promoter and the essential Gli motif in the ES enhancer. Importantly, overexpression of Zic1 and Pax3 in the 10T1/2 mesodermal cell model results in enrichment of these factors at the endogenous Myf5 locus and induction of Myf5 expression. In our previous work, we showed that by enhancing nuclear translocation of Gli factors, Zics provide spatiotemporal patterning for Gli family members in the epaxial induction of Myf5 expression. Our current study indicates a complementary mechanism in which association with DNA-bound Pax3 strengthens the ability of both Zic1 and Gli2 to transactivate Myf5 in the epaxial somite.

The folate metabolic enzyme ALDH1L1 is restricted to the midline of the early CNS, suggesting a role in human neural tube defects.

Folate supplementation prevents up to 70% of human neural tube defects (NTDs), although the precise cellular and metabolic sites of action remain undefined. One possibility is that folate modulates the function of metabolic enzymes expressed in cellular populations involved in neural tube closure. Here we show that the folate metabolic enzyme ALDH1L1 is cell-specifically expressed in PAX3-negative radial glia at the midline of the neural tube during early murine embryogenesis. Midline restriction is not a general property of this branch of folate metabolism, as MTHFD1 displays broad and apparently ubiquitous expression throughout the neural tube. Consistent with previous work showing antiproliferative effects in vitro, ALDH1L1 upregulation during central nervous system (CNS) development correlates with reduced proliferation and most midline ALDH1L1(+) cells are quiescent. These data provide the first evidence for localized differences in folate metabolism within the early neural tube and suggest that folate might modulate proliferation via effects on midline Aldh1l1(+) cells. To begin addressing its role in neurulation, we analyzed a microdeletion mouse strain lacking Aldh1l1 and observed neither increased failure of neural tube closure nor detectable proliferation defects. Although these results indicate that loss-of-function Aldh1l1 mutations do not impair these processes in mice, the specific midline expression of ALDH1L1 and its ability to dominantly suppress proliferation in a folate responsive manner may suggest that mutations contributing to disease are gain-of-function, rather than loss-of-function. Moreover, a role for loss-of-function mutations in human NTDs remains possible, as Mthfr null mice do not develop NTDs even though MTHFR mutations increase human NTD risk.

Impact of methylenetetrahydrofolate reductase deficiency and low dietary folate on the development of neural tube defects in splotch mice.

BACKGROUND: The etiology of neural tube defects (NTDs) is multifactorial, with environmental and genetic determinants. Folate supplementation prevents the majority of NTDs, and a polymorphism in methylenetetrahydrofolate reductase (MTHFR) has become recognized as a genetic risk factor. The mechanisms by which folate affects NTD development are unclear. The Splotch (Sp) mouse is a well-characterized mouse model for studying spontaneous NTDs. To assess the potential interaction between folate metabolism and the Sp mutant in NTD development, we studied mice with both Sp and Mthfr mutations, as well as the interaction between Sp and low dietary folate. METHODS: Wild-type, single Mthfr+/-mutant, single Sp/+mutant, and double mutant (Mthfr+/-, Sp/+) female mice were mated with males of the same genotype. Embryos were examined for NTDs on gestational day (GD) 13.5. To investigate the effects of folate deficiency on Sp mice, Sp/+female mice were fed a control diet (CD), a moderately folic acid-deficient diet (MFADD), or a severely folic acid-deficient diet (SFADD). They were mated with Sp/+males and the embryos were examined. RESULTS: There were no differences in the incidence or severity of NTDs in embryos from double-mutant mating pairs compared to those from single Sp mutants. Embryos from Mthfr+/-dams did not exhibit NTDs. Diets deficient in folate did not influence the incidence or severity of NTDs in embryos from Sp/+mice. CONCLUSIONS: We did not observe an interaction between Sp and Mthfr mutations, or between the Sp mutation and low dietary folate, in NTD development in Splotch mice.

Spontaneous neural tube defects in splotch mice supplemented with selected micronutrients.

Splotch (Sp/Sp) mice homozygous for a mutation in the Pax3 gene inevitably present with neural tube defects (NTDs), along with other associated congenital anomalies. The affected mutant embryos usually die by gestation days (E) 12-13. In the present study, the effect of modifier genes from a new genetic background (CXL-Sp) and periconceptional supplementation with selected micronutrients (folic acid, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, methionine, myoinositol, thiamine, thymidine, and alpha-tocopherol) was determined with respect to the incidence of NTDs. In order to explore how different exposure parameters (time, dose, and route of compound administration) modulate the beneficial effects of micronutrient supplementation, female mice received either short- or long-term nutrient supplements via enteral or parenteral routes. Embryos were collected on E12.5 and examined for the presence of anterior or posterior NTDs. Additionally, whole mount in situ hybridization studies were conducted in order to reveal/confirm normal expression patterns of the Pax3 gene during neurulation in the wild-type and Sp/Sp homozygous mutant mouse embryos utilized in this study. A strong Pax3 signal was demonstrated in CXL-Sp embryos during neural tube closure (E9.5 to E10.5). The intensity and spatial pattern of expression were similar to other Splotch mutant mice. Of all the micronutrients tested, only supplementation with folic acid or 5-methyltetrahydrofolate rescued the normal phenotype in Sp/Sp embryos. When the folate supplementation dose was increased to 200 mg/kg in the diet, the incidence of rescued splotch homozygotes reached 30%; however, this was accompanied by six-fold increased resorption rate.

Developmental consequences of abnormal folate transport during murine heart morphogenesis.

BACKGROUND: Folic acid is essential for the synthesis of nucleotides and methyl transfer reactions. Folic acid-binding protein one (Folbp1) is the primary mediator of folic acid transport into murine cells. Folbp1 knockout mouse embryos die in utero with multiple malformations, including severe congenital heart defects (CHDs). Although maternal folate supplementation is believed to prevent human conotruncal heart defects, its precise role during cardiac morphogenesis remains unclear. In this study, we examined the role of folic acid on the phenotypic expression of heart defects in Folbp1 mice, mindful of the importance of neural crest cells to the formation of the conotruncus. METHODS: To determine if the Folbp1 gene participates in the commitment and differentiation of the cardiomyocytes, relative levels of dead and proliferating precursor cells in the heart were examined by flow cytometry, Western blot, and immunohistostaining. RESULTS: Our studies revealed that impaired folic acid transport results in extensive apoptosis-mediated cell death, which concentrated in the interventricular septum and truncus arteriosus, thus being anatomically restricted to the two regions of congenital heart defects. Together with a reduced proliferative capacity of the cardiomyocytes, the limited size of the available precursor cell pool may contribute to the observed cardiac defects. Notably, there is a substantial reduction in Pax-3 expression in the region of the presumptive migrating cardiac neural crest, suggesting that this cell population may be the most severely affected by the massive cell death. CONCLUSIONS: Our findings demonstrate for the first time a prominent role of the Folbp1 gene in mediating susceptibility to heart defects.

Disruption of Meox or Gli activity ablates skeletal myogenesis in P19 cells.

Gli2 and Meox1 are transcription factors that are expressed in the developing somite and play roles in the commitment of cells to the skeletal muscle lineage. To further define their roles in regulating myogenesis, the function of wild type and dominant-negative forms of Gli2 and Meox1 were examined in the context of differentiating P19 stem cells. We found that Gli2 overexpression up-regulated transcript levels of Meox1 and, conversely, Meox1 overexpression resulted in the upregulation of Gli2 transcripts. Furthermore, dominant-negative forms of either Meox1 or Gli2 disrupted the ability of P19 cells to commit to the muscle lineage and to properly express either Gli2 or Meox1, respectively. Finally, Pax3 transcripts were induced by Gli2 overexpression and lost in the presence of either mutants Meox1 or Gli2. Taken together, these results support the existence of a regulatory loop between Gli2, Meox1, and Pax3 that is essential for specification of mesodermal cells into the muscle lineage.

C/EBP-delta induction by gp130 signaling. Role in transition to myelin gene expressing phenotype in a melanoma cell line model.

Expression of genes encoding structural myelin proteins marks the inception of the myelinating Schwann cell (SC) phenotype. Earlier embryonic SC as well as adult non-myelinating SC produce the intermediate filament glial fibrillary acid protein (GFAP), which disappears from the myelinating SC. We previously observed that triggering of the gp130 receptor system by the IL6RIL6 ligand, comprising interleukin-6 (IL-6) fused to the soluble IL-6 receptor, induces myelin gene expression in rat embryonic dorsal root ganglia (DRG) cultures as well as in the murine melanoma cell line B16/F10.9. Study of target genes regulated by IL6RIL6 indicates a strong and selective induction of the transcriptional regulator C/EBP-delta in DRG cultures and in the F10.9 cell line. As shown here, silencing of C/EBP-delta mRNA and protein expression by introduction of small interference RNA-producing plasmids in the F10.9 cells prevented the induction of myelin protein zero (P0) and myelin basic protein (MBP) mRNAs by IL6RIL6. Doxycycline-regulated overexpression of C/EBP-delta was sufficient to induce accumulation of P0 and MBP mRNAs, the effect being selective, because C/EBP-delta did not affect several other genes strongly regulated by IL6RIL6. Interestingly, GFAP was inhibited by C/EBP-delta overexpression, leading to a modulation of the ratio between myelin gene products versus GFAP and suggesting that C/EBP-delta plays a role in the switch to a myelinating phenotype. The down-regulation of Pax3, also typical of the transition to myelinating cells, was observed after C/EBP-delta expression in correlation to P0 induction and to decrease of melanogenesis and cell growth. In cultures of dissociated cells of embryonic rat DRG, where we knocked-down the C/EBP-delta mRNA, we found an inhibition of P0 mRNA induction by IL6RIL6, showing that the role of C/EBP-delta on this myelin gene is not unique to the melanoma system.

Effects of folate supplementation on the risk of spontaneous and induced neural tube defects in Splotch mice.

BACKGROUND: Neural tube defects (NTDs) are among the most common human congenital malformations. Although clinical investigations have reported that periconceptional folic acid supplementation can reduce the occurrence of these defects, its mechanism remains unknown. Therefore, the murine mutant Splotch, which has a high incidence of spontaneous NTDs, along with the inbred strains SWV and LM/Bc, were used to investigate the relationship between folate and NTDs. METHODS: To investigate whether folates could reduce spontaneous NTDs, heterozygous Splotch dams (+/Sp) were treated with either folate or folinic acid throughout neurulation, gestational day (GD) 6.5 to 10.5. On GD 18.5 the dams were sacrificed and the fetuses examined for any neural tube defects. Subsequently, Sp/+ dams were treated with arsenic while receiving either a folate or folinic acid supplementation. Similar experiments were performed in the LM/Bc and SWV strains. RESULTS: Neither folate nor folinic acid supplements reduced the frequency of spontaneous NTDs in the embryos from Splotch heterozygote crosses. Arsenic increased the frequency of NTDs and embryonic death in the Splotch, LM/Bc and SWV litters and folinic acid failed to ameliorate the teratogenic effect of this metal. A folate supplement given to arsenic-treated dams proved to be maternally lethal in all three strains. CONCLUSIONS: Splotch embryos were not protected from either spontaneous or arsenic-induced NTDs by folinic or folic acid supplementation. Furthermore, folinic acid supplements did not reduce the incidence of arsenic-induced NTDs in either the LM/Bc or SWV litters.

Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates proliferation, dissociation, migration and morphogenesis of cells in culture. To investigate a possible role for HGF/SF and its receptor, the Met tyrosine kinase, in embryonic development, we have analyzed their expression in mouse embryos from day 7.5 of gestation by whole-mount in situ hybridization. Met expression is first detected in the ventral portion of somites at day 9.25 of gestation (22 somite embryo) at the level of fore limb buds. As somites mature, met expression is detected in caudal somites, and is confined to the lateral and media] tips of the dermomyotome and dermomyotome/myotome respectively. In contrast, HGF/SF is expressed exclusively in the mesodermal core of the limb bud. As the dermomyotome elongates ventrolaterally, the met-expressing cells at the lateral tip appear to detach from the somite, invade the limb bud and localize at the dorsal and ventral limb sides in close proximity to HGF/SF-expressing cells. At later stages, both met- and HGF/SF-expressing cells appear to migrate distally and localize to the digit forming area of the developing hand plate. Met expression in the lateral dermomyotome and limb bud coincides with expression of Pax-3, a marker for migrating muscle precursor cells in the somite and limb. Splotch-2H and Splotch-delayed mice, which harbor mutations in Pax-3, show major disruptions in early limb muscle development. Significantly, no met-expressing cells were observed in the limbs of homozygous Splotch-2H and Splotch-delayed animals, whereas HGF/SF expression was not affected. The restricted expression of met to a sub-population of Pax-3-expressing cells in the lateral tip of the dermomyotome, demonstrates that met represents a unique molecular marker for this migratory cell population. From these observations, together with the biological activities of HGF/SF, we propose that in homozygous Splotch embryos the failure of muscle precursors to migrate into and populate the limb bud results from a loss of met expression in the cells at the ventrolateral edge of the somitic dermomyotome.