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, Each marker represents a single zebrafish embryo, n = 8-10 embryos per group. Black bar is the mean, error bars represent SEM, and * indicates p<0.05, two-tailed Student's t-test, ns-not significant. ROI-region of interest. (D) Same samples as in C plotted for the PAX3-FOXO1 injection groups only. (E) Quantification of TUNEL-positive pixels normalized to GFP-positive pixels indicated a lower proportion of PAX3-FOXO1 cells are undergoing apoptosis in the context of the tp53 M214K/M214K mutation. Black bar is the mean, error bars represent SEM, n = 8-10 embryos per group, * indicates p<0.05, two-tailed Student's t-test. (F) Same samples as in E plotted for the PAX3-FOXO1 injection groups only. (G) Representative images from wildtype and tp53 M214K/M214K uninjected controls, CMV-GFP2A injection controls, CMV-GFP2A-PAX3 and CMV-GFP2A-PAX3FOXO1, GFP-PAX3FOXO1 developmental expression. (B) Survival of PAX3 injected wildtype (n = 89) or tp53 M214K/M214K (n = 146) as compared to PAX3-FOXO1 injected wildtype (n = 199) or tp53 M214K/M214K (n = 219) evaluated at 6, 24, 48, and 72 hr post fertilization

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, Zebrafish embryos were injected at the single-cell stage with the beta actin promoter driving GFP-Cherry, GFP-PAX3FOXO1, mCherry-HES3, or combined mCherry-HES3 and GFP-PAX3FOXO1. Shown are representative embryos at 24 hr post fertilization with indicated transgene expression. (B) Representative overlays of zebrafish embryo musculature that were fixed at 24 hr and immunofluorescence performed for myosin (red) and, Figure 4. HES3 inhibits myogenic differentiation in developing zebrafish and supports persistence of PAX3-FOXO1-positive cells. (A)

, SD is derived from technical triplicates. * indicates significant differences between treatment group and the GFP-mCherry control at a threshold of p<0.05, two-tailed Student's t-test. (D) Representative overlay of images from co-injections of mCherry-HES3 and GFP-PAX3FOXO1 from the same embryo at 24 and 72 hr post-fertilization. Images were taken with the same exposure settings and objective. (E) Quantification of the number of positive pixels for each embryo imaged at 24 and 72 hr post-fertilization. GFP-positive pixels are plotted after the same settings are applied for imaging and analysis. Each marker represents a single zebrafish embryo at 24 or 72 hr post fertilization, n = 6-12 embryos per group. Black bar is the mean, and * indicates p<0.05, two-tailed Student's t-test. ns-not significant. (F) Same analysis as in E but for mCherry positive pixels, = 5 embryos were harvested at 24 hr and markers of myogenesis assessed by qRT-PCR, including myod, myog, myl1, and myhz2