Fig. 3. Loss of Smad4 from the epiblast disrupts midline formation but does not perturb mesoderm development. (A-E) Developmental time course of (A-E) wild-type (WT) and (A'-E') Sox2Cre;Smad4^CA/N (mut) embryos. (A,A') At early gastrulation (E6.5), wild-type and mutant embryos are indistinguishable. (B,B') By E7.0, mutant embryos have a broadened embryonic region that becomes progressively distorted as development continues. (C,C') At the early headfold stage (E7.75), anterior elaboration of neural folds and invagination of foregut do not occur in the mutant. (D,D',E,E') By E8.5, the mutant embryo has developed within a normal visceral yolk sac (vys). Spherical head fold-like structures appear (hf), a protruding heart (ht)-like structure is observed anteriorly, and somites (so) form, which are fused across the midline. fb, forebrain; nt, neural tube. (F-K) Transverse sections of (F,H,J) wild-type and (G,I,K) E7.5 mutant embryos. (H) Anteriorly, the wild-type embryo shows the normal topological arrangement of the three definitive germ layers: inner anterior ectoderm or epiblast (ep); mesodermal (m) wings; and outer layer of definitive endoderm (de). At the midline, the epiblast directly contacts the axial mesendoderm (ame; red arrow) without an intervening layer of mesoderm. Arrowheads indicate parietal endoderm. (I) In mutant embryos, however, interposing mesoderm (m; red arrow) is observed at the midline, and (J,K) the posterior primitive streak (ps) is broadened compared with the control embryos. (L) Sagittal section of an E8.5 Sox2Cre/+;Smad4^CA/N embryo reveals a rudimentary heart (ht), neural tissue approximating a headfold (hf) as well as visceral yolk sac endoderm with associated blood islands (vys + bl). am, amnion. (M-O) Coronal sections of a similarly staged E8.5 mutant embryo at (M) anterior, (N) mid and (O) posterior levels. The neural plate (np) fails to form the neural tube. Somitic (so) and lateral plate mesoderm (lpm) is seen. Posteriorly, stalk-like chorionic tissue (ch) extends from the amnion and is associated with allantois (al). (P) PCR performed on microdissected tissue fragments of embryos of a Smad4^CA/CA x Sox2Cre;Smad4^N/+ intercross. In embryos expressing the Sox2Cre transgene (lanes 2-4, 7, 8), conversion of the conditional allele to the null allele is seen. The yolk sac, which is derived from the epiblast and extra-embryonic endoderm, shows only partial conversion. The epiblast-derived allantois and headfold tissue show complete conditional-to-null conversion.