Alcohol Exposure Negatively Affects Cranial Neural Crest Development In Early Chick Embryo

ObjectivesExcess alcohol consumption during pregnancy could potentially be teratogenic for the developing fetus,leading to a condition called fetal alcohol syndrome(FAS).However,the molecular mechanism leading to craniofacial abnormality,a feature of FAS,is still poorly understood.The cranial neural crest cells(NCCs)contribute to craniofacial formatio n,hence the development of these cells in the presence of ethanol was investigated-using chick embryos and in vitro explant culture as experimental models.MethodsWe demonstrated that exposure to 2% ethanol for 14 days induced craniofacial defects in the developing chick fetus,using alizarin red staining for the presence of bones.HH1 staged embryos were incubated in EC culture in the presence of 0.719% simple saline(control)or 2% ethanol until the embryo reached 10 somite stage(HH10).Utilized technics such as in situ hybridization,reverse transcription quantitative polymerase chain reaction(RT-qPCR),analyzed the expression of the key genes in the procedure of the cranial neural crest cells(CNCCs)and adherens junction genes in cranial neural crest epithelial-mesenchymal transition(EMT)process to investigate ethanol affecting neural crest development.Meanwhile,using cranial neural tube explant verified the results on chick embryos.ResultsIt was determined that exposing early chick embryos to ethanol(for 14 days)significantly increased the incidence of parietal bone defects and premaxilla shortening as compared with saline-treated(control)embryos.Immunofluorescent staining revealed that ethanol treatment repressed Ap-2?,Pax7 and HNK-1 expression by cranial NCCs,the progenitor cells of the craniofacial bones.Using double-immunofluorescent stainings for Ap-2? plus p HIS3 and Ap-2? plus Caspasese3,we showed that ethanol treatment inhibited cranial NCC proliferation and increased apoptosis.Moreover,ethanol treatment of the dorsal neuroepithelium increased Laminin,N-Cadherin and Cadherin 6B expression while Cadherin 7 expression was repressed.In situ hybridization also showed that ethanol up-regulated Cadherin 6B expression but down-regulated slug,Msx1,FoxD3 and BMP4 expression.ConclusionIn summary,our experimental results revealed that ethanol treatment interferes with the production of cranial NCCs by affecting the cell proliferation and apoptosis of these cells.In addition,it affected the delamination,EMT and cell migration of cranial NCCs,which may have contributed to the development of the craniofacial defects.