title: Retinoic acid promotes expression of germline-specific genes in chicken blastoderm cells by stimulating Smad1/5 phosphorylation in a feeder-free culture system.
abstract: Producing transgenic chickens with chicken blastodermal cells (cBCs) is inefficient due to the extremely low germline transmission capacity of cBCs. As chicken primordial germ cells (PGCs) have been reported as an efficient method for producing transgenic chickens, the inefficiency of cBCs could potentially be resolved by inducing them to differentiate into germ cells. However, whether chemical inducers are able to enhance cBCs germline competence in vitro is unknown and the molecular mechanisms of differentiation of chicken pluripotent cells into germ cells are poorly understood.
  
  
  
  We cultured cBCs with a monolayer morphology in E8 medium, a xeno- and feeder-free medium. We showed that retinoic acid (RA) treatment increased expression of germ cell-specific genes in cBCs. Using western blot, we determined that RA stimulated Smad1/5 phosphorylation. Moreover, Smad1/5 activation regulates the expression of germ cell-specific genes, as co-treatment with a Smad1/5 phosphorylation inhibitor or activator alters expression of these genes. We also demonstrate that Smad1/5 is required for RA-induced differentiation by RNA interference knockdown.
  
  
  
  Our results demonstrated that E8 medium is able to maintain cBC growth for weeks and RA treatment induced germ cell differentiation of cBCs through the BMP-Smad1/5 signaling pathway.
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
authors.forename: Xiaochuan
authors.initials: X
authors.lastname: Tang
authors.affiliation: College of Animal Science and Technology, Jingling Institute of Technology, Nanjing, 210095, People's Republic of China.
authors.forename: Shiyong
authors.initials: S
authors.lastname: Xu
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
authors.forename: Hongpeng
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Jingling Institute of Technology, Nanjing, 210095, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
authors.forename: Rongyang
authors.initials: R
authors.lastname: Li
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
authors.forename: Wangjun
authors.initials: W
authors.lastname: Wu
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
authors.forename: Minli
authors.initials: M
authors.lastname: Yu
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China. liuhonglin@njau.edu.cn.
authors.forename: Honglin
authors.initials: H
authors.lastname: Liu
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D001756:Blastoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051898:Smad1 Protein / Q000378:metabolism*; D051902:Smad5 Protein / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D051898:Smad1 Protein; D051902:Smad5 Protein; D014212:Tretinoin
keywords: Chicken blastodermal cells; E8 medium; Germ cell differentiation; Growth factor; Retinoic acid; Smad1/5 phosphorylation