BMP signaling is lost when and where DVE is newly formed. The DVE region was monitored by detection of the expression of a Hex-Venus transgene (blue) before (A) or at the indicated times after (C–E) removal of ExE. Wild-type (A, F, and I) or Cerl^−/− (H) embryos or wild-type embryo explants stripped of ExE (C–E, G, and J) at E5.5 were subjected to immunohistofluorescence staining with antibodies to p-Smad1 (pS1; A and C–H, green) or to p-Smad2 (pS2; I and J, green). Merged images with nuclear staining (Nuc; red) are also shown. Green color in merged images indicates increased level of p-Smad1 or p-Smad2 staining. The explants shown in G and J were examined after culture for 6 h. Arrowheads in C–E indicate the boundaries of Hex-Venus expression. The experimental strategy for explant experiments is shown in B. The ExE was separated from the epiblast and embryonic VE by a cut along the embryonic–extraembryonic junction at E5.5. The epiblast and embryonic VE were then cultured alone. The effects of ExE removal on expression of Hex-Venus (K) and p-Smad1 staining (L) at the indicated times of culture are summarized. Blue and orange bars indicate no change and expansion of the Venus positive domain (K) or no change and down-regulation of p-Smad1 (L), respectively. The numbers of embryos showing each pattern are indicated. Bars, 50 µm.