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Verification of chicken Nanog as an epiblast marker and identification of chicken PouV as Pou5f3 by newly raised antibodies
Author(s) -
Nakanoh Shota,
Fuse Naoyuki,
Takahashi Yoshiko,
Agata Kiyokazu
Publication year - 2015
Publication title -
development, growth and differentiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 66
eISSN - 1440-169X
pISSN - 0012-1592
DOI - 10.1111/dgd.12205
Subject(s) - homeobox protein nanog , epiblast , embryonic stem cell , biology , antibody , embryoid body , polyclonal antibodies , microbiology and biotechnology , nanog homeobox protein , stem cell , induced pluripotent stem cell , immunostaining , sox2 , immunohistochemistry , embryo , immunology , embryogenesis , gastrulation , genetics , gene
Pluripotency is an important feature of early embryonic cells of multicellular organisms. Recent advances in stem cell research have shown that Nanog and Pou5f1 (Oct3/4) play important roles in mammalian pluripotency. However, whether these molecules exert conserved functions in other species remains unknown. Although the epiblast of the early chicken embryo would provide a useful experimental model, a lack of antibodies against chicken Nanog ( cN anog) and chicken PouV/Pou5f3 ( cP ouV) proteins has hampered intensive investigation. Here we report newly raised polyclonal antibodies that specifically recognize cN anog and cP ouV proteins. The specificity and sensitivity of the antibodies were validated by both western blotting and immunostaining with transfected 293T cells and chicken embryonic tissues. Immunohistochemistry using these antibodies revealed that cN anog protein was specifically localized in epiblastic cells and germ cells. In contrast, cP ouV expression was seen almost ubiquitously. We also found that chicken epiblast‐derived colony‐forming cells that morphologically resemble mouse embryonic stem cells were cN anog‐positive, implying that these colony‐forming cells possess pluripotency. The anti‐ cP ouV antibody further enabled us to identify a previously unknown region at the N‐terminus of the cP ouV protein containing a characteristic motif that is absent in mammalian Pou5f1. Thus, the antibodies raised in this study are useful tools for studying the functions of cN anog and cP ouV at the protein level and the molecular mechanisms of chicken pluripotency.