Embryonic development and analysis of cell death in wild-type and mdm2^-/- embryos. Embryos were obtained from an incross of wild-type or mdm2^+/- zebrafish. (a) Brightfield images of wild-type and mdm2^-/-embryos at matching developmental stages show the lethal phenotype due to the homozygous loss of mdm2. (b) The 24-hpf wild-type and mdm2^-/- embryos were stained live with acridine orange and imaged under ultraviloet light. Increased green fluorescence in mdm2^-/- embryos compared with wild-type embryos suggests the presence of more apoptotic cells. (c) Whole-cell lysate was prepared from a number of wild-type/mdm2^+/- and mdm2^-/- embryos and used in a Caspase 3/7 assay. Cells from mdm2^-/- embryos have higher Caspase 3/7 activity compared with wild-type/mdm2^+/- siblings.

Rescue of mdm2^-/- embryos with microinjection of FLAG-mdm2 mRNA. Single-cell embryos collected from an incross of mdm2^+/- fish were injected with 25 or 50 pg of mRNA and imaged at ~30 hpf. (a) Wild type/mdm2^+/- sibling embryo; (b) uninjected mdm2^-/-embryo; (c) mdm2^-/- embryo injected with 25 pg FLAG-mdm2 mRNA; (d) mdm2^-/- embryo injected with 50 pg FLAG-mdm2 mRNA; (e) mdm2^-/- embryo injected with 25 pg FLAG-mdm2C448A mRNA; and (f) mdm2^-/- embryo injected with 50 pg FLAG-mdm2C448A mRNA.

mdm2^-/- embryos accumulate high levels of p53 protein and have increased p53 target gene transcription. (a) Whole mount immunohistochemistry was performed on embryos from an incross of mdm2^+/- fish. The embryos were fixed in methanol:acetone (1:1) at the six-somite stage and stained with p53-5.1 hybridoma supernatant (green) and DAPI counterstain (blue). The stained embryos were deyolked, dorsally mounted in glycerol and imaged at the trunk at a × 40 magnification on a confocal microscope. (b) Western blot analysis on lysates of embryos from different incrosses—wild type, mdm2^Δ15/15, mdm2^+/-, p53^M214K/M214K and mdm2^-/-; p53^M214K/M214K. A unit of 20 Gy gamma irradiation was performed on 24 hpf embryos and the total lysate from all embryos was collected at 28 hpf. A unit of 30 µg of lysate was loaded in each lane of the SDS-PAGE gel. (c) Quantitative real-time PCR analysis of p53 target genes in wild-type and mutant mdm2 embryos. RNA was extracted from 50 embryos at 24 hpf in TRIzol, and purified before utilizing 1 µg of total RNA in a reverse transcription reaction. A volume of 0.5 µl of each cDNA was used in a quantitative real-time PCR reaction set up in triplicate in a 384-well plate. The gene expression data was analyzed using actin and EF1a as internal controls. *P<0.05 for Student’s t-test.

mdm2^-/-; p53^M214K/M214K embryos accumulate mutant p53 protein. Whole mount immunohistochemistry was performed on mdm2^-/-; p53^M214K/M214K and wild-type embryos at 30 hpf. The embryos were fixed in methanol:acetone (1:1) and stained with p53-5.1 hybridoma supernatant (green) and DAPI counterstain (blue). The stained embryos were deyolked, dorsally mounted in glycerol and imaged at the midbrain at × 40 magnification using a confocal microscope. (a, b) Magnified insets showing different mutant p53 localization in different cells in the mdm2^-/-; p53^M214K/M214K embryo.

mdm2^-/-; p53^M214K/M214K adult zebrafish accumulate mutant p53 protein in most tissues. Five-month-old mdm2^-/-; p53^M214K/M214K, mdm4^-/-; p53^M214K/M214K and p53^M214K/M214K adult zebrafish were sacrificed, fixed and embedded in paraffin for histological analysis with the p53-5.1 hybridoma supernatant. The sections were counterstained with hematoxylin and imaged under brightfield at × 40 magnification. E, epidermis; CE, columnar epithelium; CSM, circular smooth muscle; T, renal tubule; H, hepatocytes; S, sinusoid

Accumulation of mutant p53 protein in tumors of p53^M214K/M214K fish. Adult p53^M214K/M214K zebrafish that were observed to have developed tumors in the eye and trunk were sacrificed, fixed and embedded in paraffin for histological analysis with the p53-5.1 hybridoma supernatant. The sections were counterstained with hematoxylin and imaged under brightfield. (a) Normal eye from a p53^M214K/M214K fish ( × 10 magnification). (b) Tumor in the eye of a p53^M214K/M214K fish ( × 10 magnification). (c) Tumor in the eye of a p53^M214K/M214K fish ( × 40 magnification). (d) Tumor in the trunk of a p53^M214K/M214K fish ( × 40 magnification). P, pigmentation of eye lens.

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