Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development

Abstract

We report here the characterization of a mutant mouse line with a specific gene trap event in the Mdm4 locus. Absence of Mdm4 expression results in embryonic lethality (10.5 days postcoitum [dpc]), which was rescued by transferring the Mdm4 mutation into a Trp53-null background. Mutant embryos were characterized by overall growth deficiency, anemia, improper neural tube closure, and dilation of lateral ventricles. In situ analysis demonstrated increased levels of p21(CIP1/Waf1) and lower levels of Cyclin E and proliferating cell nuclear antigen expression. Consistent with lack of 5-bromo-2'-deoxyuridine incorporation, these data suggest a block of mutant embryo cells in the G(1) phase of the cell cycle. Accordingly, Mdm4-deficient mouse embryonic fibroblasts manifested a greatly reduced proliferative capacity in culture. Moreover, extensive p53-dependent cell death was specifically detected in the developing central nervous system of the Mdm4 mutant embryos. These findings unambiguously assign a critical role for Mdm4 as a negative regulator of p53 and suggest that Mdm4 could contribute to neoplasias retaining wild-type Trp53. Finally, we provide evidence indicating that Mdm4 plays no role on cell proliferation or cell cycle control that is distinct from its ability to modulate p53 function.

FIG.1.

A gene trap event in the Mdm4 locus leads to a null Mdm4 mutation and early embryonic lethality in mice. (A) The structure and size of the retroviral construct along with the structures of the wild-type and mutated alleles and the positions of the oligonucleotides used for the PCR-based strategy are depicted. Exons (black vertical bars) and the long terminal repeats (LTRs) of the viral vector (white boxes) are indicated. SA and SD are the splicing acceptor site and donor site, respectively; neo^r, neomycin resistance gene. (B) Mdm4 wild-type and mutant embryos (homozygous) at different stages of embryonic development (E10.5 and E11.5). (C) Mdm4/Trp53 wild-type and mutant (heterozygous and homozygous) embryos at different stages of embryonic development (E10.5 and E12.5). (D) PCR analysis of embryos with the indicated genotypes. The positions of the three primers a, b, and c are indicated. (E) Western blotting analysis of Mdm4 protein expression. NIH 3T3 cells (as a control) or mouse brain protein extracts from wild-type (Mdm4^+/+ Trp53^+/+) or mutant (Mdm4^+/− Trp53^+/+, Mdm4^+/− Trp53^+/−, and Mdm4^−/− Trp53^−/−) embryos were immunoprecipitated with the anti-Mdm4 rabbit polyclonal antibodies p55, p56, and SI23 (anti-X [α-X]) or with preimmune (PI) serum. Mdm4 was detected by Western blot analysis using a mixture of monoclonal antibodies 6B1A and 11F4D.

FIG. 2.

ISH analysis of an E11.5 mouse embryo with an Mdm4 antisense probe. Abbreviations: F, forebrain; M, midbrain; Hi, hindbrain; H, heart.

FIG. 3.

Impaired cellular proliferation in Mdm4 mutant embryos. (A and B) Decreased Cyclin E1 expression in Mdm4^−/− embryos compared to wild-type littermates as determined by ISH analysis; the whole embryos (A) and close-up of the fetal livers (B) are shown. (C) Analysis of proliferation of the fetal liver hematopoietic progenitors. Embryos were stained with an antibody to detect in vivo BrdU incorporation and counterstained using hematoxylin and eosin. Very few BrdU-positive cells are found in the fetal liver of Mdm4 homozygous mutant embryos. (D) Increased p21 expression in Mdm4^−/− embryos compared to wild-type littermates as determined by ISH analysis.

FIG. 4.

(A to D) Impaired proliferation of Mdm4-null fibroblasts in cultures. (A) Growth curves for Mdm4^+/+, Mdm4^+/−, Mdm4^−/−, and Mdm4^−/− Trp53^−/− MEF cultures at passage 2. A total of 2.5 × 10 ⁴ cells were plated into 12-well plates. Cultures were harvested at daily intervals,and the total number of cells were determined and normalized to the number of cells at day 0 (20 h after plating) (relative number [Nb] of cells). The numbers refer to the mean values of two independently derived MEF cultures of each genotype. (B) Level of BrdU incorporation by asynchronously growing passage 2 Mdm4^+/− Trp53^+/− and Mdm4^−/− Trp53^+/− MEFs. (C) Quantitative analysis of the percentage of S-phase cells after serum stimulation for 24 h of G ₀-synchronized Mdm4^+/+, Mdm4^+/−, and Mdm4^−/− MEF cultures. The BrdU-labeled MEFs were analyzed by indirect immunofluorescence, and the S-phase cells were determined by staining with anti-BrdU antibody. (D) Photomicrographs of MEFs at passage 2. Mdm4-null MEFs were growth arrested; Mdm4^+/+ MEFs were not growth arrested. (E) Constitutive high levels of p21 protein expression in Mdm4^−/− MEFs at passage 1. Western blotting analysis from Mdm4^+/+ cell lysates, two independent Mdm4^+/−, and Mdm4^−/− MEF cultures at passage 1. Vinculin serves as loading control. (F and G) Constitutive high levels of p53 protein expression in Mdm4^−/− MEFs at passage 3. (F) p53 protein levels determined by Western blotting analysis using the sheep polyclonal antibody Ab-7 from Mdm4^+/− and Mdm4^−/− MEF cell lysates. Vinculin serves as a loading control. (G) p53 protein (red) levels examined by indirect immunofluorescence using the rabbit polyclonal antibody CM5 in Mdm4^+/− and Mdm4^−/− MEF cultures. The DNA (blue) is stained with 4′,6′-diamidino-2-phenylindole (Dapi). The absence of signal in Mdm4^+/− Trp53^−/− MEF cultures demonstrates the specificity of the antibody.

FIG. 5.

(A and B) Proliferation of embryonic fibroblasts (A) Growth rates of fibroblasts that are either wild type, heterozygous, or nullizygous for Mdm4 and nullizygous for p53. Cell numbers at each point represent an average of two separate lines of cells, two plates of cells for each line. (B) Colony formation of wild-type, heterozygous, or nullizygous for Mdm4 and p53 MEFs grown in conditions of low cell density (10³ cells per 10-cm-diameter plate). Values given represent an average of two plates each of two lines of cells. Bars indicating standard deviation values are shown. A total of 2 × 10 ³ cells were plated (10-cm-diameter dishes), cultured, fixed, and stained. Representative dishes are shown over the bars for the corresponding genotypes. (C) Colony formation following DNA damage. Wild-type MEFs or MEFs heterozygous or nullizygous for Mdm4 and p53 were plated at low density (10⁴ cells per 10-cm-diameter dish) following treatment with increasing UV irradiation (2, 5, and 10 J/m²). Two independent experiments were performed, and the mean values with standard deviations (error bars) are presented. (D, E, and F) Cell cycle analysis on embryonic fibroblasts determined by FACScan analysis. The S-phase cells (BrdU-positive cells) are revealed by staining with anti-BrdU antibody, and DNA content is revealed with propidium iodide. (D) G₁/S ratio of MEFs that are wild type, heterozygous, or nullizygous for Mdm4 and p53. (E) Effects of gamma irradiation on the cell cycles of MEFs that are wild type, heterozygous, or nullizygous for Mdm4 and p53. The G₁/S ratio is given compared to untreated G₁/S values. (F) S-phase entry following serum stimulation and gamma irradiation of synchronized MEFs that are wild type, heterozygous, or nullizygous for Mdm4 and p53. The percentages of cells in the S phase of the cell cycle immediately after starvation, cells grown for 24 h in growth medium (not treated [NT]), or cells that had undergone gamma irradiation (X-Ray).

FIG. 6.

(A) Analysis of proliferation, apoptosis, and p21 expression levels in the developing CNS (hindbrain and spinal cord) of Mdm4^−/− and Mdm4^+/+ embryos 12 days postcoitum. We stained wild-type and Mdm4 mutant embryos with antibody recognizing PCNA to detect cell proliferation and with antibody directed against the activated form of caspase 3 to detect apoptosis. The p21 expression pattern in the same regions of the CNS was determined by ISH. Dark field (red signal) view of sections counterstained with Hoechst (blue signal). Positive cells are indicated by the arrows. vl, ventricular lumen. (B) Absence of apoptosis in the developing CNS of E11.5 Mdm4^−/− p53^−/− embryos. Mdm4^+/+ p53^+/−, Mdm4^−/− p53^+/−, Mdm4^−/− p53^−/−, and Mdm4^+/+ p53^−/− embryos were stained with antibody directed against the activated form of caspase 3 to detect apoptosis. The ventricular zone (vz) is indicated.