Fig. 4. Loss of anterior primitive streak derivatives in Smad2^+/–;Smad3^–/– mutant embryos. Whole-mount in situ hybridization of (A,C,E,G,I,K,K',M,O,S, U,W,Y) wild-type (WT) and (B,D,F,H,J,L,L',N,P,T,V,X,Z) Smad2^+/–;Smad3^–/– mutant (mut) embryos at (A,B,Q) E7.25, (C-J,R) E7.5, (M-P) E7.75 and (K-L,S-Z) E8.5. (A-F) Foxa2 transcripts first identify the AVE (red line), then AME cells emerging from the anterior streak and later demarcate the node (n). (A,B) Foxa2 and (Q) Cer1 expression in the AVE confirms the normal establishment of the primary anteroposterior axis. However, mutant embryos show few (B) to no (D,F) Foxa2-expressing AME cells extending anteriorly. Similar results were obtained with Shh (G-J) that is also diagnostic for the AME and node. Note that Foxa2 expression is retained in the patent node (D,F). In both control and mutant embryos at the early somite stage (K-L), Shh expression persists in the node and identifies its derivative the notochord (nc). At the early headfold stage, (M,N) Nog and (O,P) Chrd mark the node, midline and posterior PCP (bracket), and are downregulated and truncated anteriorly in mutant embryos (N,P). Note the expanded anterior neuroectoderm in N,P, and lack of neural groove in P. (Q,R) Lower levels of the nascent definitive endoderm markers Cer1 and Hhex are observed during early gastrulation. At E8.5, Hhex marks involuting foregut endoderm and lateral angioblasts (ab) (S). Foregut Hhex expression is absent, but retained in angioblasts in mutant embryos (T). Similarly, Shh expression in the hindgut (hg) endoderm of mutants is largely lost (K',L'). (U-X) The loss of anterior structures is revealed by diminished Six3 and Otx2 expression. (Y,Z) Fgf8 is expressed in the anterior neural ridge (arrowhead) and mid/hindbrain boundary (asterisk) in the wild type. In mutant embryos, anteriormost Fgf8 transcripts are absent, whereas expression in the mid/hindbrain region is normal (Z). (K,L) Hindbrain formation is unperturbed as assessed by Krox20 expression in rhombomeres 3 and 5. (W-Z) Posterior T expression indicates ongoing gastrulation.