Compared to eutherian mammals, birds retain a primitive form of epiblast development. Molecular studies of the avian epiblast can provide valuable insight for mammalian epiblast research. Here, we introduce several basic techniques in handling epiblast-stage embryos of the chick, the major model organism for avian developmental biology studies. We describe how to collect embryos for RNA extraction and gene expression analysis, to set up ex ovo New culture for overexpression, bead graft and small molecule-based inhibitor studies, and to carry out whole-mount RNA in situ hybridization analysis. We introduce a novel and simple method for molecular perturbation of the epiblast differentiation in ovo. We also describe how to perform primary chicken epiblast cell culture, to establish stable epiblast stem cell (Epi-SC) lines, and to assay for pluripotency in primary epiblast cells and Epi-SCs.
