EWS-FLI1 induces SRBCTs in zebrafish. (A) Schematic of Tol2 transposons including the hsp70 or the β-actin promoter, FLAG-tag (black rectangle), human EWS-FLI1 coding sequence, IRES-GFP sequence (gray rectangle), flanked by Tol2 recombination sites (triangles). (B-H) H&E staining of sagittal sections of adult zebrafish. (B) Control adult zebrafish. Wild-type zebrafish injected with EWS-FLI1 developed an invasive leukemia like-tumor (C magnified in D) or a solid small round blue cell tumor of the eye (arrow in E) (E magnified in F). tp53 mutants injected with EWS-FLI1 transposons also develop leukemia (G) or solid tumors (H) with similar histology. Scale bars: 200 μm (B,C,E), 50 μm (D,F–H).

H&E staining of selected serially transplanted tumors originating from two different zebrafish SRBCTs. (A) Tumor D85; (B) tumor D86 (see supplementary material Table S1). Numbers indicate serial transplants (primary, secondary, tertiary etc.).

Gene expression in zebrafish SRBCTs is distinct from MPNSTs and similar to human EWS-FLI1 gene expression data. (A) Histology of representative zebrafish MPNST (left) and SRBCT (right) that were used for microarray analysis. Scale bars: 50 μm. (B) Heat map of 421 probes that exhibited differential expression between MPNSTs and SRBCTs. Yellow, high expression; blue; low expression. (C) Quantitative RT-PCR of selected SRBCT upregulated genes. (D,E) GSEA using human homologs of zebrafish genes shown in B compared with human gene expression data from (D) stable knockdown of EWS-FLI1 (red, higher expression in A673 Ewing’s cell line; blue, higher expression following EWS-FLI1 knockdown in A673 cells) or (E) inducible expression of EWS-FLI1 in a bone-marrow-derived stromal cell line (red, higher expression in EWS-FLI1 expressing cells; blue, higher expression in control cells).

EWS-FLI1 induces developmental defects in zebrafish embryos. (A) Non-transgenic (GFP–) embryos are wild type. (B) Transgenic embryos (GFP+) carrying a heat-shock-inducible EWS-FLI1 transgene exhibit pericardial edema (arrow) and reduced head and eye size. (C) A morpholino against the transgene decreases the penetrance of the pericardial edema phenotype. (D) Percentage of transgenic embryos exhibiting pericardial edema is decreased using a morpholino targeting the EWS-FLI1 transgene. (E) RT-PCR for EWS-FLI1 shows low level of expression without heat shock (–HS) and increased expression following heat shock (+HS). no RT, no RT negative control.

EWS-FLI1 affects convergence and extension in zebrafish embryos. Heat-shocked control (A) and EWS-FLI1 transgenic (B) embryos at 2 d.p.f. reveals that transgenic embryos are shorter with reduced head development. At the 18 somite stage, in situ hybridization for myoD in heat-shocked control (C) and EWS-FLI1 transgenic (D) reveals a wider somite width (E) and a shorter anterior-posterior axis as measured by the angle between the head and tail (F) in transgenic embryos (*P<0.02, **P<0.001; error bars indicate s.e.m.).

Abnormal mitotic spindles in hsp70:EWS-FLI1 embryos. Wild type (A-F) and Tg(hps70:EWS-FLI1) embryos (G-L) were heat shocked at the 50% epiboly stage and allowed to develop to 24 hpf before being fixed and stained with an antibody to alpha-tubulin (green) and with DAPI (nuclei, blue). Individual mitotic cells from the trunk and tail of 24 hpf embryos are shown.