Fig. 6. Arrested development of yolk sac vasculature in Tbx20 null embryos. (A,B) LacZ expression in yolk sac flat mounts showing lack of mature vessels in Tbx20^lacZ/lacZ embryos, as seen in wild type. (C,D) Sections of E9.5 Tbx20^lacZ/+ or Tbx20^lacZ/lacZ yolk sacs showing expression of lacZ in the mesodermal layer, but not in blood cells. (E,F) Tbx20 is expressed in yolk sac mesoderm and vessel derivatives of Tbx20^lacZ/+ embryos from E11.5-14.5. Note that expression was never observed in the endodermal layer or hematopoietic cells. (G,H) Whole-mount immunohistochemical detection of Pecam1, a marker of mature endothelial cells at E9.5. (I,J) TUNEL showing increased apoptosis in the mesodermal layer of E9.5 mutant yolk sacs compared with wild type. (K,L) Transmission electron microscopy of E9.5 yolk sacs showing an example of perforation of the endothelial layer (red arrowheads) in the mutant. (M) RT-PCR analysis of E9.0 yolk sacs (n=3) for markers of hemangioblast specification (Tal1), angioblast specification (Kdr) and remodeling (Angpt1, Tek) and vessel maturation (Acta2, Gja5). Scale bar: 5 µm. e, endoderm; ec, endothelial; wt, wild type.