Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo

Abstract

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.

Role of FGF signalling in neural crest cell migration during early chick embryo development

“…The eggs were incubated until the chick embryos reached the desired developmental stage. For gene transfection, 0.5 μl of plasmid DNAs (1.5 mg/ml green fluorescent protein (GFP), Sprouty2-GFP or Dn-FGFR1-GFP) (Yang et al, 2002a;Wang et al, 2013Wang et al, , 2015Wang et al, , 2017 were microinjected into the neural tubes of HH10 (Hamburger and Hamilton stage 10) chick embryos (Hamburger and Hamilton, 1951). Using a pair of platinum wires fashioned in parallel as electrodes, the plasmids carrying the gene of interest were transfected randomly into the neural tube cells by electroporation.…”

Augmented BMP signaling commits cranial neural crest cells to a chondrogenic fate by suppressing autophagic β-catenin degradation

“…It has been reported that autophagy induced by ciliation directs human embryonic stem cells to a neuroectoderm lineage by degrading the fate determinant (28). In neural crest cells, autophagy is known to be involved in regulating their generation, survival, and differentiation into neurons in vitro (29,30). However, it remains unclear whether functional coordination between BMP and autophagy contributes to the regulation of stem cell fate, especially CNCCs in the context of craniofacial development.…”

Cell survival controlled by lens‐derived Sema3A–Nrp1 is vital on caffeine‐suppressed corneal innervation during chick organogenesis

“…Whole-mount embryos, the cornea of the elder stage chicken embryos, embryo sections and SH-SY5Y cells were stained using antibodies against neurofilament (NF), phospho-Histone3 (pHIS3), Ki67, C-caspase3, or Slit2 as previously described (Ma et al, 2014;Wang et al, 2017). Briefly, the specimens were incubated with monoclonal antibody: Mouse anti-NF Invitrogen) secondary antibodies.…”

“…SH-SY5Y was collected and lysed with CytoBuster™ Protein Extraction Reagent (#71009, Novagen, Beijing, China). The western blot analysis was performed according to the previously described (Wang et al, 2017). The antibodies used were Ephb2 (1:1,000; Invitrogen) and β-actin (1:3,000; Proteintech, Rosemont, IL).…”