Fig. 6. Rescue of blood island formation by wild-type visceral endoderm. (A) Schematic representation of the aggregation setup: tetraploid Vegf wild-type (wt) GFP+ embryos were aggregated with diploid Vegflo/lo embryos (F2), resulting in chimaeric visceral endoderm and Vegflo/lo mesoderm. (B) Whole-mount photograph of a 9.0 dpc embryo and its chimaeric yolk sac (ys), which was taken under GFP visualising light conditions. The black arrowhead points to trace contribution of tetraploid cells to the gut epithelium. White arrowheads indicate the remains of the yolk sac attached to the embryo. (C) One of the GFP-positive patches in the yolk sac. (D) Photograph of the same area as in C using light conditions that allow the visualisation of GFP-negative cells as well. (E) The same area as in C,D after lacZ staining. (F) Appropriate (green arrow) and impaired (blue arrow) blood island formation in the chimaeric yolk sac. Higher magnifications of the respective areas are shown in G,H. (I) ß-galactosidase staining reveals blood islands only in areas where the visceral endoderm is Vegf wild type (lacZ-negative cells, green arrows), whereas blood islands cannot be formed in opposition to Vegflo/lo (lacZ-positive cells, blue arrows) endoderm. (L) Higher magnification of the boxed area in I. The short range rescue of blood island formation by VEGFA secreted from wild-type visceral endoderm is marked by an asterisk. (J,K) Extensive wild-type tetraploid contribution to the yolk sac visceral endoderm is not sufficient to rescue intra-embryonic vascular defects. Lumina of the dorsal aortae (black arrows) are barely detectable (J). An increase in the number of blood cells (in the heart in K and sinus venosus in J (red arrows) is observed with increasing wild-type tetraploid contribution. h, heart; sv, sinus venosus.
