Synergistic roles of Mdm2 and Mdm4 for p53 inhibition in central nervous system development

Abstract

Loss of Mdm2 or Mdm4 leads to embryo lethal phenotypes that are p53-dependent. To determine whether Mdm2 and Mdm4 inhibit p53 function redundantly in a more restricted cell type, conditional alleles were crossed to a neuronal specific Cre transgene to delete Mdm2 and Mdm4 in the CNS. Mice lacking Mdm2 in the CNS developed hydranencephaly at embryonic day 12.5 due to apoptosis, whereas Mdm4 deletion showed a proencephaly phenotype at embryonic day 17.5 because of cell cycle arrest and apoptosis. The deletion of both genes, strikingly, contributed to an even earlier and more severe CNS phenotype. Additionally, Mdm2 and Mdm4 had a gene dosage effect, because loss of three of the four Mdm alleles also showed a more accelerated CNS phenotype than deletion of either gene alone. All phenotypes were rescued by deletion of p53. Thus, these in vivo data demonstrate the importance of Mdm4 independent of Mdm2 in inhibition of p53.

Fig. 1.

Hydranencephaly in mice lacking Mdm2 in the CNS. (A) Whole-mount LacZ staining of E12.5 embryos. (B) Hematoxylin/eosin (H&E)-stained sections of the forebrain at E12.5. (C) Embryos at E14.5. (D) Sagittal sections of embryos stained with H&E at E14.5. (E) Cross sections of E18.5 embryos. All embryos have the Nes-Cre transgene. The other alleles are labeled as follows: M2^FM/+, Mdm2^FM/+, M2^FM/FM, Mdm2^FM/FM, and M2^−/FM, Mdm2^−/FM; VZ, ventricular zone; IZ, intermediate zone; MZ, marginal zone; V, ventricle; LV, lateral ventricle; and C, cortex.

Fig. 2.

Proencephaly in mice lacking Mdm4 in the CNS. (A) Specific recombination of the Mdm4^FX allele in E14.5 Mdm4^FX/FX Nes-Cre embryos. Numbers label Mdm4 exons (boxes). The closed circle is an frt site, and diamonds are loxP sites. The triangles are PCR primers used for detection of the recombination event. F4 and E2re amplify the Mdm4 conditional allele. F4 and In2re2 amplify the Mdm4 allele (Δ2) after recombination. Hematoxylin/eosin-stained sagittal (B) and coronal (C) sections of embryos at E18.5. All embryos have the Nes-Cre transgene. M4^FX/+, Mdm4^FX/+, M4^FX/FX, and Mdm4^FX/FX; M, 1-kb-plus marker; In, intestine; Lu, Lung; Li, Liver; Tl, Tail; Br, Brain; H, Heart; C, cortex; V, ventricle; and LV, lateral ventricle.

Fig. 3.

Apoptosis and proliferation in mice lacking Mdm2 or Mdm4 in the forebrain. TUNEL (A and C) and BrdUrd assays (B and D) in the CNS from E10.5 embryonic brains (A and B) from control Mdm2^FM/+ Nes-Cre (M2^FM/+) and mutant Mdm2^FM/FM Nes-Cre (M2^FM/FM) mice. E14.5 embryonic brains (C and D) from control Mdm4^FX/+ Nes-Cre (M4^FX/+) and mutant Mdm4^FX/FX Nes-Cre (M4^FX/FX) mice. VZ, ventricular zone; IZ, intermediate zone; SP, subplate; and V, ventricle. (Scale bars, 50 μm for A and B; 100 μm for C and D.)

Fig. 4.

Loss of Mdm2 and Mdm4 caused increased p53 protein levels. p53 immunohistochemical staining of E10.5 (A and B) and E14.5 (C) sections of the forebrain. (D) p21 immunohistochemical staining of E14.5 embryonic brains. (E) Western blot analysis of embryo extracts. Real-time RT-PCR of Mdm2^FM/FM Nes-Cre (F) and Mdm4^FX/FX Nes-Cre (G) samples. V, ventricle; VZ, ventricular zone; IZ, intermediate zone; and SP, subplate. (Scale bars, 50 μm for A and B; 100 μm for C and D.)

Fig. 5.

Loss of Mdm2 and Mdm4 in the CNS caused an earlier and more severe phenotype than loss of either gene alone. Embryos and hematoxylin/eosin sections at E11.5 (A and B) and E13.5 (C and D) Arrows indicate hemorrhaging in the brain. Embryos at E14.5 were stained for LacZ and shown as whole embryos (E) and cross sections (F). M2, Mdm2; M4, Mdm4; D, dorsal; V, ventral; and FP, floor plate.

Fig. 6.

Apoptosis and cell cycle arrest in embryos lacking Mdm2 and Mdm4 in the CNS. BrdUrd (A), TUNEL (B), and p21 immunohistochemical staining (C) of CNS sections of E10.5 embryos. (D) Real-time RT-PCR was performed by using RNA from heads of E10.5 wild-type (Ctl), Mdm2^FM/FM Nes-Cre (M2), Mdm4^FX/FX Nes-Cre (M4), and Mdm2^FM/FM Mdm4^FX/FX Nes-Cre (DN) embryos. V, ventricle. (Scale bars, 100 μm.)