Fig. 5. Definitive endoderm is not formed in Smad4 mutant embryos. (A,B) Hex transcripts mark the anterior visceral endoderm (AVE) and emerging definitive endoderm (DE) at the early primitive streak stage. Smad4 mutant embryos express Hex in the AVE, but not in the ADE. (C-F) A Hex-lacZ transgene identifies a portion of medial DE cells (C, lateral view; D, frontal view) at E7.5. By contrast, (E, lateral view; F, frontal view) no transgene activity is detected in mutant embryos. (G,H) Cerl expression within the DE shows a drastic reduction in distribution and intensity in mutant embryos, while expression in the AVE is retained. (I,J) At E7.5 Foxa2 transcripts mark the axial mesendoderm that extends from the anterior streak to the embryonic/extra-embryonic junction. Smad4 mutant embryos show residual Foxa2 expression with the AVE but lack axial expression. (K,L) Mixl1, a marker of the primitive streak and a putative downstream target of Smad4, is correctly expressed in mutants at the late primitive streak stage. (M-P) lacZ expression in (M,N) control and (O,P) Sox2Cre/+;Smad4^CA/N;ROSA26^R/+ E7.5 embryos viewed in coronal section. In control embryos, the definitive endoderm layer (arrows) is blue, indicating an epiblast-derived origin. By contrast, this layer is white in mutant embryos, indicating origin from non epiblast-derived visceral endoderm and failure to form DE. Note the absence of midline (m) in mutant embryos. Arrowheads indicate parietal endoderm.