Figure 2. Clonal down-regulation of Tead4 expression and TE-differentiation inhibition.

A schematic of experimental strategy to effect clonal Tead4 knockdown (KD) and TE-inhibition in one-half of the embryo using microinjected RDBs ± Tead4-dsRNA (see materials and methods) is given on the left. Representative single z-plane confocal micrographs of control and Tead4-KD embryos at either the mid-16-cell (E3.1), 32-cell (E3.6) or >64-cell (E4.5) stages immuno-stained for Tead4 or Cdx2 (green) are given. Cells derived from the microinjected 2-cell stage clone are distinguishable by the co-injected RDB fluorescence (red). DNA counter-stain (blue) is also shown. In merged images the arrows denote cells exhibiting a lack of Tead4 or Cdx2 expression in the Tead4-KD microinjected cell clone, thus confirming the efficacy and the functional and clonal inhibition of TE-differentiation by Tead4-dsRNA until the late blastocyst stage (E4.5). ICM cells not from the microinjected clone, expressing Tead4 protein are marked with asterisks (in Tead4 alone micrographs). Note, in contrast to global Tead4-KD embryos (Fig. 1), such clonal Tead4-KD embryos initiate blastocoel formation in a manner indistinguishable from control microinjected embryos. Scale bars = 10 μm.