Figure 3.

The polar trophectoderm induces cup-shape formation of the epiblast

(A) Correlation analysis of polar TE aspect ratio (total height/length of interface) with curvature. Stages II–V had a strong positive correlation. Stage I, n = 68; stage II, n = 81; stage III n = 51; stage IV, n = 40; stage V = 44. Analysis: r = 0.7414, p < 0.0001. Black, stage I; red, stage II; yellow, stage III; blue, stage IV; green, stage V.

(B) Correlation analysis of polar TE aspect ratio with the length of the tissue interface. Strong anti-correlation of stages II–V. Stage I, n = 68; stage II, n = 81; stage III, n = 51; stage IV, n = 40; stage V, n = 44. Analysis: r = −0.7572, p < 0.0001.

(C) Staining of E4.5 embryo cultured for 48 h in hanging drops after immuno-surgery (left and middle columns) and control (right column). Embryos stained for DAPI (red), F-actin (green), and HNF4alpha (blue, top row); Otx2 in the bottom row.

(D) Quantification of the circularity of the epiblast after immuno-surgery and hanging drop culture. For treated embryos (IS), only those were analyzed where the full TE tissue could be removed. For controls (Ctrl), only embryos that retained all three lineages were considered. Treated, n = 6; control, n = 4. Scatterplot, mean ± SEM.

(E) Mouse embryonic stem cells (mESCs) were cultured for 48 h in 3D Matrigel in differentiating conditions. Structures stained for DAPI (red), F-actin (green), and Otx2 (white).

(F) Quantitative analysis of the circularity of mESC structures. n = 40. Scatterplot, mean ± SEM. Structures were collected from 4 independent experiments.

(G) Model for the hypothetical forces required to regulate EPIBLAST cup-shape acquisition. Blue, polar TE; magenta-purple, epiblast. Scale bars, 20 μm.