Hypoxia disrupts erythropoietic islands. (A,B) The effect of hypoxia on erythropoietic island numbers and numbers of erythroblasts per macrophage was quantified as described previously (Figure 3E,F). Hypoxia had a greater effect on both island integrity and numbers of erythroblasts per macrophage than did the Rb status of the cells. Differences in numbers of erythroblasts per macrophage as a function of oxygen tension (21% vs 1% O₂) were significant in the absence of Cre expression (P < .001) and in the presence of Cre expression (P < .001). The numbers of islands formed at 21% compared with 1% O₂ were also significant (P < .04 for Cre− and P < .02 for Cre+). Error bars represent standard deviation from the mean value for numbers of erythroblasts per island (A) and numbers of islands (B). (C-H) The effect of hypoxia on erythroblast island integrity was visualized by staining of adherent (C-F) and floating (G,H) cells from purified island cultures exposed to 21% oxygen for 36 hours (C,D) or to 21% oxygen for 18 hours and subsequently to 1.0% oxygen for 18 hours (E-H). Significant differences were observed for the number of erythroblasts per macrophage in E13.5 wild-type and Rb-null fetal liver after 4 hours (P = .001) or 16 hours (P = .001) in culture at 21% oxygen and between Rb-positive and Rb-deficient cultures derived from Rb^flox mice when cultured at 1% oxygen versus 21% oxygen for 12 hours (P = .001 for Cre+; P = .001 for Cre−) but not between wild type and Rb null earlier in gestation or possessing a wild-type placenta (Rb^flox;Cre+ vs Cre−). See ″Fluorescence microscopy and island counts″ for image acquisition information. (B) Island numbers were reduced in E13.5 Rb-null fetal liver compared with wild type (P = .01) and in conditionally targeted fetal livers following 16 hours at 1% oxygen when compared with normal tissue culture (P = .02 and P = .04, respectively) Notably, placental rescue increased Rb-deficient island number (P = .03; Student t test).