Loss of suz12 in p53/nf1-deficient fish diversifies carcinogenesis. (A) CRISPR-Cas9-mediated targeting truncates Suz12a and Suz12b proteins before their functional domains, conferring a loss of function. VEFS, VRN2-EMF2-FIS2-SUZ12 domain; WDB, WD-40 binding domain; Zn, Zn-finger region. (B) p53/nf1/suz12-deficient fish are prone to tumors in various anatomical locations, e.g. the abdomen or head (arrows). (C) Histopathology analysis reveals a variety of cancer types in p53/nf1/suz12-deficient tumor-bearing fish, whereas p53/nf1-deficient control fish solely develop MPNSTs. Numbers underneath representative images indicate the frequency of the respective tumor type in nf1a^+/− or nf1^+/+ tumor-bearing fish analyzed by histology. Scale bars: 100 µm.

Tumor watch of suz12-mutant fish in the p53^m/m, nf1b^−/−, nf1a^+/− andp53^m/m, nf1b^−/−, nf1a+/+ backgrounds. Representative experiments are displayed to show tumor onset time for fish with nf1a^+/− genotype (A) or nf1a^+/+ genotype (B), plotted by suz12 genotype; either three out of four (suz12^{+/−/−/−}; left), two out of four (suz12^+/+/−/−; middle), or one out of four (suz12^+/+/+/−; right) suz12 alleles are knocked out. The data from each knockout population was compared to the respective suz12-wild-type control population to calculate P-values using Student's t-tests.

Observation of multiple tumor foci in p53/nf1/suz12-deficient zebrafish lines. (A) Fluorescence microscopy image of an adult p53^+/−, nf1b^−/−, nf1a^+/−, suz12a^+/−, suz12b^+/− fish bearing two independent GFP-positive tumors in abdominal and anal locations. (B) Proportion of fish with multiple lesions in different suz12-mutant populations with additional p53^+/−, nf1b^−/−, nf1a^+/− or p53^+/−, nf1b^−/−, nf1a^+/+ backgrounds. Data are mean±s.d. of populations with any combination of three out of four (suz12^{+/−/−/−}), two out of four (suz12^+/+/−/−), or one out of four (suz12^+/+/+/−) suz12 alleles knocked out. The total number of fish analyzed is indicated (n). P-values were calculated using Student's t-tests. (C) Representative HE-stained sections of p53/nf1/suz12-deficient fish with two separate tumor foci. Squares indicate areas shown in magnified images below.

Analysis of H3K27me3 and pan-cytokeratin by indirect immunofluorescence and confocal microscopy. Paraffin-embedded sections of the indicated tumor types from zebrafish with the genotypes indicated were stained for H3K27me3 (green) and pan-cytokeratin (panCK; red). DNA is visualized using Hoechst 33342 (blue). Scale bars: 10 µm.

Evaluation and inhibition of the Ras signaling pathway in suz12-mutant and suz12-wild-type MPNSTs. (A) Immunohistochemistry analysis of signaling in three individual suz12-mutant (suz12^{+/−/−/−}) and suz12-wild-type (suz12^+/+/+/+) MPNSTs (n=3) each stained for phosphorylation of ERK, AKT, and S6 (p-ERK, p-AKT, and p-S6), indicating activation of Ras signaling. (B) The suz12a^+/−, suz12b^−/− (mut) or suz12-wild-type control MPNST (wt) tumor cell growth in the pericardial cavity of implanted embryos. These embryos were treated with DMSO vehicle control or PD-3025901 (PD901; 25 or 50 nM) (n=9 fish per treatment, doses based on the maximum tolerated dose of the individual drug). The fluorescent tumor area was determined for each embryo at 3 dpf (pre-treatment) and 7 dpf (post-treatment), and was reported as the normalized ratio of the red fluorescent area at 3  dpf versus 7 dpf in individual embryos. Individual values with medians (black bars) are shown. *P<0.05, **P=0.0064, ***P<0.0001 (Student's t-test). (C) Representative fish images at 3 dpf and 7 dpf after DMSO control or 50 nM PD0325901 treatment.