Fig. 4

Tissue boundary is necessary for polar trophectoderm expansion. a Implanted blastocyst (E4.75) stained for phosphorylated ERK (pERK). pERK is detected in the primitive endoderm (PE) and the trophectoderm (TE). pERK is restricted to the polar region of the TE. b Implanted blastocyst (E4.75) stained for phosphorylated AKT (pAKT). pAKT is restricted to the polar region of the TE. For a and b, representative example of 10 embryos. c Schematic representation of the experimental design to examine the contribution of actomyosin contractility and FGF signalling in the formation of polar/mural TE tissue boundary formation. d Representative examples of control, Y27632 (ROCK inhibitor) and SU5402 (Fgfr inhibitor) treated blastocysts cultured as described in c and analysed for the presence of polar/mural TE boundary (cyan dotted line). Polar/mural tissue boundary formation is defective in the absence of actomyosin contractility and FGF signalling. e Quantification of polar/mural TE boundary formation efficiency in control, Y27632 and SU5402-treated blastocysts. χ² test; ****P < 0.0001, mean ± SEM. For d and e FGF n = 29 control, 15 Y27632 treated and 15 SU5402-treated embryos. f Quantification of cell–cell junction angles at the polar/mural TE interface. The angles of junctions after abrogation of actomyosin contractility (Y27632) and signalling (SU5402) are narrowed in agreement with the defective formation of polar/mural TE tissue boundary in these conditions. Two-sided unpaired student’s t test; ****P < 0.0001; mean ± SEM. Control: n = 57; Y27632: n = 38; SU5402: n = 45. g Representative examples of control, Y27632 (ROCK inhibitor) and SU5402 (FGFR inhibitor)-treated blastocyst cultured as described in b and analysed for polar TE (white outline) expansion. h Quantification polar TE expansion efficiency in control, Y27632, and SU5402-treated blastocysts. χ² test; ****P < 0.0001. For f and g n = 29 control, 15 Y27632 treated and 15 SU5402-treated embryos. i Model for the molecular and cellular events necessary for polar TE expansion during peri-implantation morphogenesis. Source data are provided as a Source Data file. Scale bars = 20 µm