23As a neonicotinoid pesticide, imidacloprid is widely used to control sucking 24 insects on agricultural planting and fleas on domestic animals. However, the extent to 25 which imidacloprid exposure has an influence on cardiogensis in early embryogenesis 26 is still poorly understood. In vertebrates, the heart is the first organ to be formed. In 27 this study to address whether or not imidacloprid exposure affects early heart 28 development, the early chick embryo has been used as an experimental model because

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7 neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7 and HNK-1 cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1 neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.