Fig. 2.

Pum1^−/−; Pum2^−/− mice are developmentally delayed and embryonic lethal by e8.5. Immunofluorescence staining of wild-type and mutant embryos for Oct4 (red) and endoderm marker Gata4 (green). (A and B) The e3.5 mutant embryos have no (or a smaller) blastocoel cavity and lack Gata4-positive primitive endoderm (PrE) cells. (C and D) The e4.5 mutant embryos have randomly positioned PrE cells in the ICM. (E and F) The e5.5 wild-type embryos develop an inner layer of Oct4-positive epiblast cells and an outer layer of visceral Gata4-positive endoderm cells. In mutant embryos, Gata4-positive PrE cells remain next to the blastocoel cavity. Nuclei were stained with DAPI (blue). (Scale bars, A–F, 25 µm.) (G) The e8.5 embryonic and extraembryonic tissues from Pum1^+/−; Pum2^+/− mated mice. Yolk sacs were used for genotyping. A Pum1^−/−; Pum2^−/− embryo (far right) is smaller than all littermates. (Scale bar, 1 mm.) (H–K) Higher magnification of dissected e8.5 embryos; the Pum1^−/−; Pum2^−/− double-knockout embryo (Inset in J, magnified in K) shows developmental delay, a primitive head fold, and overall lack of tissue with especially thin neural tissue. (Scale bars, H–K, 500 µm.)