FIG. 2.

Fasudil treatment phenocopies the Y-27632-induced disruption of the ICM morphology. Blastocysts were cultured in the absence (control) or presence of 5 μM Fasudil from E3.5 to E4.5 and immunostained for cell lineage-specific transcription factors. A) GATA4-positive PrE (red) and NANOG-positive Epi (green) cells are less tightly packed together and are more spread out in the Fasudil-treated embryo than in the control embryo. Confocal images are z-series projections. B) Three-dimensional reconstructions of confocal microscopic images of the ICM of embryos in A. Reconstructed three-dimensional images are rotated to show views from the PrE and Epi side of the ICM. C) Comparison of ICM size based on the widest length of ICM cell dispersion. Circles represent the ICM size in individual embryos, and horizontal bars represent the mean value for each group. The ICM is significantly wider (Student t-test) in inhibitor-treated embryos (n = 12) than in the control embryos (n = 14). D) Comparison of total cell numbers. Graph shows the mean and standard error. There is no statistically significant difference (P = 0.87; Student t-test) between control (n = 14) and inhibitor-treated (n = 14) embryos. E) Comparison of the number of PrE (red bar) and Epi (green bar) cells shows no statistically significant differences (P[PrE] = 0.16, P[Epi] = 0.29; Student t-test) between control (n = 14) and inhibitor-treated (n = 14) embryos. Graph shows the mean and standard error. Data presented in C, D, and E are compilations of two replicates of the experiment that were performed on different occasions. Blue, DAPI. Bars = 20 μm (A, B).