Fig. 4. ICMs lacking Mbd3 fail to expand pluripotent cells in vivo and in vitro. (A) Embryos in which implantation was delayed by diapause were collected after 3 days of diapause (See Materials and methods) and stained for Oct4 (white), Nanog (green) and Gata4 (red). Nuclei were stained with DAPI (blue). Genotype of embryos is indicated in the phase contrast images. (B) ICMs from Mbd3^+/- (top panels) or Mbd3^-/- (middle and bottom panels) 3.5 dpc embryos were isolated using immunosurgery and allowed to attach and outgrow in the presence of LIF for 3 days. Pictures were taken every 24 hours. (C) Number and morphology of ICM outgrowths after 3 days in culture. Any outgrowths with a cell mass in addition to monolayer endoderm were scored as having `cell mass+endoderm' irrespective of the size of the cell mass. Outgrowths with adherent cells only were classed as `endoderm only'. Outgrowths containing fewer than 10 cells after 3 days were classed as `<10 cells'. The number of outgrowths of each genotype is indicated (N). (D) Cell masses isolated from ICM outgrowths cultured for 5 days were stained for Oct4 (green). Genotyopes are indicated in each panel.