Fig. 4. Nodal signals are required for definitive endoderm specification. (A-E) Nodal^600/– mutant embryos at 7.5 dpc (A,B) and 8.5 dpc (C-E). Hematoxylin and Eosin stained sections of the embryos are shown in the bottom of each panel. Two classes of embryo are apparent by 7.5 dpc (Table 1); those that gastrulate inside the visceral yolk sac (VYS) (A), and those that gastrulate externally (B). Twenty-four hours later, embryos remaining inside the VYS form an AP axis, but show defects in heart looping and anterior truncations (C). Embryos that grow external to the VYS develop an AP axis with somites along the trunk; however, fused somites indicate the midline may be defective in many embryos. The posterior is closely associated with the VYS in approximately half of these embryos (D), and in the others both the anterior and posterior are fully external to the VYS (E). end, endoderm; neur, neurectoderm; PS, primitive streak; som, somite. (F-M) Whole-mount in situ hybridization of 7.5 dpc (F,J) and 8.5 dpc (G-I,K-M) embryos. Anterior views (F,H) and lateral views with anterior towards the left (G,I-M). (F) Sonic hedgehog (Shh) is expressed in the midline anterior definitive endoderm (ADE) of WT embryos. Nodal^600/– mutant embryos show highly reduced Shh expression. (G) One day later, Shh expression in the midline extends into the ventral forebrain in the WT embryo (arrow); however, this expression domain is absent in Nodal^600/– mutant embryos. The anterior extent of the expression is indicated by the arrowhead. (H) Anterior view of the embryos shown in G, underscoring the absence of ventral forebrain expression and greatly reduced anterior foregut (fg) expression domain in the Nodal^600/– mutant embryo. (I) In severely affected Nodal^600/– mutant embryos, Shh expression is confined to the posterior midline. The embryo on the right expresses Shh along the length of the midline and in hindgut endoderm (hg). There is no obvious anterior gut endoderm population in either embryo. (J) Foxa2 is expressed in the node, midline and ADE in the WT embryo, but is highly downregulated in Nodal^600/– mutant embryos at a similar stage. (K) Later in development, Foxa2 expression in the CNS and ADE extends into the ventral forebrain of the WT embryo. By contrast, Foxa2 expression is restricted to the level of heart (indicated by arrowhead) in Nodal^600/– mutants. (L,M) Nodal^600/– mutant embryos stained with Otx2 to assess the presence of forebrain/midbrain tissue. A severely affected embryo (L) and an embryo with the less severe phenotype (M) both express Otx2 in a distinct, but highly reduced, anterior domain.