Fig. 4. The allantois undergoes normal vasculogenesis and maintains expression of Foxf1 and Bmp4 in the absence of hedgehog signaling. (A-D) Foxf1 whole-mount in situ hybridization. (A) E8.5 wt littermate of the Smo^-/- mouse embryo in D, showing Foxf1 expression in lateral mesoderm, primitive streak and allantois. (B) Foxf1 expression in lateral mesoderm, yolk sac and allantois of an E8.5 wt embryo still in the primitive, unturned position (shown for comparison with the unturned Smo^-/- embryo). (C,D) Smo^-/- embryos have the same pattern of Foxf1 expression as Ihh^-/-; Shh^-/- (see Fig. 2), i.e. in the allantois and primitive streak, but not in the lateral mesoderm or yolk sac. To show the chorioallantoic fusion, the entire E9.5 conceptus in C was left intact during hybridization. After the staining, a hole was dissected in the yolk sac through which the allantois and chorio-allantoic connection can be seen. Foxf1 is expressed throughout the allantois, which emerges from the posterior end of the embryo and spreads out like a wide funnel over the chorionic surface (inset shows the entire yolk sac at low magnification). The E8.5 Smo^-/- embryo in D has the yolk sac removed to show Foxf1 expression in the primitive streak and its absence in the lateral mesoderm. (E,F) Pecam immunostaining of allantoic explants grown in vitro. Explants of unfused allantoic buds (E8.25) were grown for 24 hours, after which the primitive vascular plexus was visualized by immunostaining for the endothelial marker Pecam. Explants from Smo^-/- embryos (F) developed a vasculature indistinguishable from that of wt embryos (E). (G-J) X-Gal staining of Bmp4^lacZ heterozygous E9.5 embryos homozygous for the wt (G,H) or null (I,J) alleles in the Smo locus. (G,I) Bmp4^lacZ expression in primitive streak and allantois is independent of hedgehog signaling, whereas expression in lateral mesoderm is lost in Smo^-/-. (H,J) Flat-mounted yolk sacs (top) and sections (bottom) showing Bmp4^lacZ expression in mesodermal cells of blood vessels. The avascular phenotype of Smo^-/- yolk sac corresponds to reduced Bmp4^lacZ expression. al, allantois; am, amnion; lm, lateral mesoderm; pl, placenta; ps, primitive streak; ysm, yolk sac mesoderm.