Figure 4.

iETX Embryos Specify Anterior and Posterior Domains on Opposite Sides

(A) E6.5 embryo with Cerl (red, arrows), Bry (green, asterisks), and Oct4 (gray). Natural embryo: 2 examples.

(B) iETX embryo at day 5 analyzed for Cerl (red, arrows), Bry (green, asterisks), and Oct4 (gray). iETX embryos: 38/69 structures, n = 4. Scale bar, 30 μm.

(C) Schematic of possible Bry/Cerl position combinations: opposite sides, same side, or Cerl is at the distal tip.

(D) Scoring iETX embryo at 5 days of development according to (C). Opposite = 33 structures, same side = 0, distal = 4, n = 4.

(E) iETX embryo at day 5 analyzed for Cerl (red, white arrows), Dkk1 (green, asterisk), Eomes (gray, yellow arrows), and DAPI (blue); max., maximum projection; 29/69, n = 3. Scale bar, 30 μm.

(F) Scoring iETX embryos at day 5 according to the position of Cerl+ve/Dkk1+ve domain in relation to Eomes. Opposite = 28, same side = 1, distal = 2, n = 3.

(G) Same as (E) but with Lefty1 (green, asterisks) and Phalloidin (blue). 18/54 examples, n = 3. Scale bar, 30 μm.

(H) Same as (F) but with Cerl/Lefty1 in relation to Eomes. Opposite = 19, same side = 1, distal = 1, n = 3.

(I) Representative iETX embryo at day 5 with ectopic Bry and analyzed for Cerl (red, white arrows), Bry (gray, yellow arrows), Oct4 (green), and Phalloidin (blue). Scale bar, 30 μm. 11/31, n = 4.

(J) Scoring iETX embryo at 5 days according to the position of Cerl in relation to the posterior domain (Bry or Eomes) as described in Figure S4D. Non-ectopic = 5, ectopic = 28, equal sides = 3, distal Cerl = 25, n = 8. (A, B, E, G, and I) Dashed rectangles are magnified on the right. Scale bar, 10 μm. (D, F, H, J) Error bars, SD. ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

See also Figure S4.