Mdm2, but not Mdm4, is required for primitive erythropoiesis. (A) Mdm2^lox/lox embryos that are either positive (EpoR^GFP-Cre/+) or negative (EpoR^+/+) for the EpoR-GFP-cre transgene at various stages of embryonic development. Mdm2^lox/lox; EpoR^GFP-Cre/+ embryos are devoid of any red cells as early as E10.5, whereas E12.5 Mdm2^lox/lox; EpoR^GFP-Cre/+;p53^−/− embryos appear normal. Scale bar equals 15 μm. (B) A representative blood island in sections of E10.5 Mdm2^lox/lox and Mdm4^lox/lox ; EpoR^GFP-Cre/+ embryos (top panels). The presence of blood cells is severely compromised in Mdm2^lox/lox mutants. Data from controls (Mdm2^lox/+ or Mdm4^lox/+; EpoR^+/+) and mutants are represented by the black and gray bars, respectively. The yolk sac sections are stained with hematoxylin and eosin. Magnification, × 20. Number of CFU-Es and monocyte-macrophage colony-forming cells (CFC-m's) detected in methylcellulose cultures supplemented with Epo or a mixture of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), respectively (bottom panels). Numbers are expressed per 5 × 10⁵ yolk sac cells. Data from controls (Mdm2^lox/+ or Mdm4^lox/+; EpoR^+/+) and mutants are represented by the black and gray bars, respectively. The data represent the means (± SD) of 6 independent experiments. There is a significant difference between the mean numbers of CFU-Es in the mdm2 mutants compared with their control littermates by the Student test (P < .001; S). (C) Mdm2 inactivation in erythroid progenitors leads to apoptotic cell death. In situ end labeling (ISEL) in blood islands of E10.5 embryos with the indicated genotypes (left panels). Note that strong background staining is observed in the visceral endoderm (en) of both control and mutant embryos. Within the blood islands, specific staining is only observed in sections from Mdm2^lox/lox; EpoR^GFP-cre/+ embryos (arrowheads). Hematoxylin and eosin–stained sections of heart from E12 fetuses (middle panels). In contrast to controls, only a few nucleated erythrocytes were found in Mdm2^lox/lox; EpoR^GFP-Cre/+ sections, and many apoptotic figures are seen (arrows). Magnification, ×100. Cleaved caspase-3 expression (casp-3*; red) in Ter119-positive erythroid progenitors (green) in the dorsal aorta of embryos with the indicated genotypes (right panels). DNA (blue) is stained with 4,6-diamidino-2-phenylindole (dapi). Magnification, ×40. (D) Mdm2 inactivation in erythroid progenitor cells leads to increased p53 protein levels. Immunohistochemistry for p53 in the blood islands (left panels) and the dorsal aorta (middle panels) and immunohistofluorescence for p53 and Ter-119 in the heart (right panels) of embryos with the indicated genotypes. p53 expression is only detected in the nuclei of most (> 90%) erythroid cells of Mdm2^lox/lox; EpoR^GFP-Cre/+ embryos. DNA (blue) is stained with dapi. Magnification, ×40.