Fig. 7
NANOG expression is diminished in Brd2tg/tg; Brd4 knockout morulae (stage 2). A Schematic illustration of the strategy for the generation of Brd2+/+; Brd4-, Brd2tg/+; Brd4-, and Brd2tg/tg; Brd4-knockout embryos with a CRISPR-Cas9 system. B–E, B’, B”, B”’, C’, C”, D’, D”, E’, E” Immunohistochemical analysis of BRD4 (B, B’, C–E; yellow), NANOG (B”, C’, D’, E’; magenta), and GATA6 protein (B”’, C”, D”, E”; green), with DAPI (nuclei, blue) staining, in Brd2+/+ (C, C’, C”), Brd2tg/+ (D, D’, D”), and Brd2tg/tg (B, B’, B”, B”’, E, E’, E”) late morulae at 16- to 31-cell stages treated with (C–E, C’, C”, D’, D”, E’, E”) or without (B, B’, B”, B”’) electroporation of CAS9 protein and Brd4 single guide (sg)RNAs. BRD4, NANOG, and GATA6 expression were normally found in Brd2tg/tg late morulae (B, B’, B”, B“’). The remaining NANOG-positive cell still expresses GATA6 (yellow arrows, E’, E”). F The ratio of numbers of NANOG- and GATA6-expressing cells to DAPI-positive cells (nuclei) in late morulae (Kruskal-Wallis test for two markers followed by Steel’s test for NANOG expression; NANOG in Brd2+/+ vs. Brd2tg/+, p = 0.092; GATA6 in Brd2+/+ vs. Brd2tg/+vs. Brd2tg/tg, p = 0.232; *p < 0.05; n.s., not significant). Red lines indicate the mean values, and green lines represent the SE bars. Individual values of marker-expressing cells are provided in Additional file 15. The sample numbers analyzed for each experiment are indicated in Additional file 19. Scale bars, 25 μm in B–E, B’, B”, B”’, C’, C”, D’, D”, E’, E”. IVF, in vitro fertilization; WT, wild type; KO, knockout