RESUMO
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.
Assuntos
Ácido Fólico/administração & dosagem , Mutação , Defeitos do Tubo Neural/etiologia , Fator de Transcrição PAX3/genética , Animais , Apoptose , Ciclo Celular/efeitos dos fármacos , Suplementos Nutricionais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Defeitos do Tubo Neural/prevenção & controle , Fator de Transcrição PAX3/fisiologiaRESUMO
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.
Assuntos
Metilação de DNA , Exposição Materna/efeitos adversos , Defeitos do Tubo Neural/genética , Fator de Transcrição PAX3/genética , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Animais , Estudos de Casos e Controles , Ilhas de CpG , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Idade Materna , Camundongos , Defeitos do Tubo Neural/induzido quimicamente , Defeitos do Tubo Neural/tratamento farmacológico , Gravidez , Regiões Promotoras Genéticas , Vitamina E/administração & dosagem , Vitamina E/farmacologiaRESUMO
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.
Assuntos
Ácido Fólico/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fenótipo , Espinha Bífida Cística/patologia , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX7/genética , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Receptor 1 de Folato/genética , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Células-Tronco Neurais/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Animais , Antagomirs/genética , Antagomirs/metabolismo , Feminino , Receptor 1 de Folato/antagonistas & inibidores , Receptor 1 de Folato/metabolismo , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/agonistas , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Transcrição PAX3/deficiência , Fator de Transcrição PAX3/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Fatores de Transcrição SOXB1/agonistas , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
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.