Fig. 2. Early Nodal signaling is required for normal epithelialization of the epiblast and to prevent ectopic expression of extra-embryonic VE markers and Furin in distal regions. (A-D) Whole-mount in-situ hybridization (ISH) of an antisense probe directed against the deleted exon 2 identifies Nodal^lacZ/lacZ null mutants (B) and reveals specific expression of Nodal mRNA in the epiblast and EmVE of wild-type embryos (A). Nodal expression is also detected by ß-galactosidase staining of Nodal^+/lacZ heterozygotes (C). In E5.25 Nodal^lacZ/lacZ homozygotes, Nodal expression independent of Nodal activity is detected only in the epiblast (D). (E-H) Immunostaining of E-cadherin (E,F) and laminin (G,H) reveals irregularities in the epithelial organization of mutant epiblasts (F), and detachment of distal embryonic visceral endoderm cells from the laminin-positive basement membrane (arrowhead in H). (I-P) In wild-type and Nodal^+/lacZ heterozygous embryos (control), Gata4, Hnf4, Ttr and Furin adopt a restricted expression in the ExVE around E5.5 (I,K,M,O). In sharp contrast, ectopic distal expression of these markers is observed in Nodal^lacZ/lacZ null mutants (J,L,N,P).