Fig. 7.

Inhibition of BET activity prolongs competency to adopt a neural progenitor state. (A) Diagram of experimental design to test the competence of IBET-treated explants to respond to a neural-inducing BMP inhibitor past the time non-treated explants normally would. (B) In situ hybridization examining the expression of sox3 and krt12.4 in explanted blastula caps treated with vehicle or IBET (250 μM) from stages 9 to 11, washed into fresh 1× MMR, treated with BMPi (K02288) (20 μM) from stages 12 to 15 and then collected for analysis. Only explants treated with IBET then BMPi were able to induce a neural fate (as evidenced by expression of sox3 but not krt12.4) similar to that of explants treated with just BMPi. (C) Western blot analysis of stage 15 explants collected at same time as explants collected for in situ analysis with the same treatment schedule. Blot was probed with anti-pSmad-1,5,8 to assess levels of active BMP signaling, and anti-actin for normalization. pSmad-1,5,8 and actin were detected on the same membrane using the same secondary antibody. (D) Diagram of experimental design to test the competence of IBET-treated explants to respond to endoderm-inducing activin past the time non-treated explants normally would. (E) In situ hybridization examining expression of endodermin in explanted blastula caps treated with vehicle or IBET from stages 9 to 10.5, then treated with activin from stages 10.5 to 12, then collected for analysis. Only explants treated with activin at stages 9 through 12 showed increased expression of endodermin. (F) Model depicting two temporally distinct roles for Brd4 in the establishment and exit from pluripotency. Scale bars: 250 μm.