Fig. 2. Defective PD to AP rotation in Nodal^600/– embryos. Primitive streak (B-D,F-I), AVE (E-I) and extra-embryonic ectoderm (K,J) marker analysis in 6.5 dpc embryos. Wild-type controls are shown above Nodal^600/–mutants. Cartoons indicate primitive streak/mesoderm markers in blue and AVE markers in pink. Anterior is towards the left. (A) Nodal^600/– embryo. Mesoderm (m) is visible proximal to the epiblast (epi), while the chorion (ch) is proximally displaced. The VE shows a distinct thickening at the distal tip (indicated by *). The embryonic/extra-embryonic boundary is indicated by the arrowhead. (B) Cripto, which is normally localized to the posterior epiblast, is expressed throughout the proximal epiblast. (C) T (brachyury) marks the nascent mesoderm and primitive streak. In Nodal^600/– mutant embryos, T is expressed symmetrically in the proximal epiblast and in the extra-embryonic mesoderm. (D) Wnt3, which is normally confined to the posterior proximal epiblast and overlying VE just prior to and following the initiation of gastrulation, is expressed symmetrically throughout the proximal epiblast in Nodal^600/– mutant embryos, but is absent from the VE (data not shown). (E) Foxa2 is expressed in the anterior primitive streak (AS) and the AVE. In Nodal^600/– mutant embryos, Foxa2 is expressed in distal VE and epiblast. (F) Hex is expressed in the AVE both prior to and following the initiation of gastrulation in wild-type embryos. Hex is also expressed in newly formed definitive endoderm (DE) by mid streak stages. Hex expression in Nodal^600/– mutant embryos is restricted to the distal most VE. (G) Lhx1 is expressed in mesoderm emerging from the anterior primitive streak (AS) and in the AVE at gastrulation, but in Nodal^600/– mutant embryos expression is present in the proximal epiblast and the distal VE. (H) Goosecoid (Gsc), a putative target of Nodal signaling, is expressed in AVE and anterior primitive streak. In Nodal^600/– mutant embryos, Gsc expression is only weakly detected in the proximal epiblast and the distal VE in a proportion of embryos and is undetectable in others. (I) Lefty1 and Lefty2 expression, examined using a common in situ hybridization probe that detects both transcripts, shows that Lefty1 (L1) is expressed in the AVE, and Lefty2 (L2) in nascent mesoderm. In Nodal^600/– mutant embryos, Lefty1 expression in the AVE is not detected. Lefty2 expression is detected in the proximal epiblast of mutant embryos. (J) The T box gene eomesodermin (Eomes) is expressed in the chorion (ch) and the posterior epiblast. In Nodal^600/– mutant embryos, expression is maintained in the chorion, while epiblast expression is proximal. (K) Expression of Bmp4, which marks extra-embryonic ectoderm, is unaffected in Nodal^600/– embryos.

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