(A) The wildtype (WT) Atrx protein consists of 2013 amino acids (aa). Targeting of the atrx coding sequence with CRISPR/Cas9 resulted in truncation of Atrx before its functional domains and confers a loss of function; numbers indicate the underlying genetic alterations, e.g., +4 = insertion of 4 bases. (B)atrx-/- embryos show developmental abnormalities and die within 18 days post fertilization (dpf); sibling includes both atrx+/- and atrx+/+ embryos.

(A) Whole-mount in situ hybridization for α-e1 and β-e1 globins in wildtype (WT), atrx+/- heterozygous fish and atrx-/- homozygous mutants at 22 hpf and 5 dpf as indicated. Boxes outline the CHT region at 5 dpf, and are magnified in the right panels. Globin signal intensities of α-e1(B) and β-e1(C) in fish with different atrx backgrounds were calculated at 5dpf. Horizontal bars indicate the means ± SEM, which were compared with the two-tailed unpaired t-test; ns = not significant; ***p<0.001. (D) Erythroid progenitors development visualized by GFP in the Tg(gata1:GFP) transgenic line with wildtype (WT) or atrx-/- background at 7 dpf. CHT = caudal hematopoietic tissue; H = heart; KM = kidney marrow; dpf = days post fertilization. (E) Analysis of peripheral blood smears by MGG staining in wildtype fish (WT), atrx-/- homozygous mutant and atrx-/- homozygous mutant injected with zebrafish atrx mRNA at 7 dpf; scale bar: 10 μm. (F) Circularity index for the erythroid cells from fish with different atrx backgrounds (E) was calculated by ImageJ. Horizontal bars indicate the means ± SEM, which were compared with the two-tailed unpaired t-test; *p<0.05; **p<0.01.

(A) The morphologic features of p53/nf1/atrx- and p53/nf1-deficient tumors were indistinguishable by visual inspection. (B) When all tumor types were considered, tumor onset was not significantly altered upon heterozygous loss of atrx; a representative tumor watch experiment using line 1 is shown; the tumor watch has been reproduced in lines 1 and 2 with similar results; ns = not significant.

This figure shows HE-stained sections of all tumor types detected in p53-/-, nf1b-/-, nf1a+/-, atrx+/- or p53-/-, nf1b-/-, nf1a+/+, atrx+/- fish that were not observed in atrx-wildtype siblings; scale bars: 50μm.

This figure shows all tumor types detected in p53-/-, nf1b-/-, nf1a+/-, atrx+/- or p53-/-, nf1b-/-, nf1a+/+, atrx+/- fish that were not observed in atrx-wildtype siblings. For each tumor a section stained by indirect immunofluorescence for H3K27me3 (green) and pan-cytokeratin (red) is shown. The DNA is visualized with Hoechst (blue); scale bars: 10μm.

(A) FPKM values show a significant downregulation of tert in p53/nf1/atrx-deficient fish compared to p53/nf1-kockout control fish; values were compared with the two-tailed unpaired t-test; n = 3, p = 0.0387. (B) Fluorescence in situ hybridization (FISH) using a Cy3-conjugated telomere-specific probe (TelC-Cy3, red) reveals elongated telomeres visible as large irregularly shaped spots in tumors with a knockout of p53, nf1 and atrx compared to atrx-wildtype control tumors, consistent with alternative lengthening of the telomeres; DNA stained with DAPI (blue); scale bars: 10μm; n = 6. (C) Quantification of relative telomere area in FISH images, normalized to DAPI signal area; values were compared with the two-tailed unpaired t-test; *p = 0.0151; n = 6. (D) Quantification of the relative number (#) of all detectable telomere spots in FISH images, normalized to DAPI signal area; values were compared with the two-tailed unpaired t-test; *p = 0.042; n = 6. (E) Quantification of the relative number (#) of telomere spots larger than ~1.5μm² in FISH images, normalized to DAPI signal area; values were compared with the two-tailed unpaired t-test; *p = 0.0013; n = 6. (F) RNA-seq analysis demonstrates significantly increased expression of PRC2-target gene sets in atrx-deficient tumors; group 1: p53-/-, nf1b-/-, nf1a+/-, atrx+/+ (control); group 2: p53-/-, nf1b-/-, nf1a+/-, atrx+/-; n = 3.