Figure 2. PAX3 and PAX3-FOXO1 have distinct impacts and tolerance during embryonic development and in adult zebrafish.

(A) Strategy for assessing functional differences of beta-actin-driven PAX3 and PAX3-FOXO1 in a vertebrate system. (B) Representative images at 24 hr post-fertilization of Uninjected, mCherry2A-PAX3, and GFP2A-PAX3FOXO1-injected zebrafish. (C) Survival curve of Uninjected, GFP2A or mCherry2A injected controls, mCherry2A-PAX3, and GFP2A-PAX3FOXO1. Error bars represent SE. Log rank test, p<0.0001 for PAX3FOXO1 versus all other conditions. (D) Embryonic phenotypes scored at 3 days post-injection. * indicates p<0.05, for PAX3 vs PAX3FOXO1, Fisher’s exact test. MO- morpholino. DV- Dorso-Ventral. (E) Percentage of GFP + cells from dissociated zebrafish embryos as quantified by fluorescent activated cell sorting (FACS). Error bars represent SD across three independent experiments. * indicates p<0.05, two-tailed Student’s t-test. (F) Adult zebrafish over 3 months of age robustly expressed beta-actin-driven Cherry, GFP, or Cherry2A-PAX3 and developed normally. Zebrafish injected with BetaActin-GFP2A-PAX3FOXO1 displayed developmental defects or developed tumors. Arrow denotes GFP + area. The percentage indicates zebrafish with detectable fluorescence at adulthood. (G) Hematoxylin and eosin staining showed normal histology of BetaActin-PAX3 expressing skeletal muscle (sagittal section) at 299 days of age, and abnormal histology of BetaActin-PAX3FOXO1 epaxial muscle exhibiting dramatic left-right asymmetry (transverse section, asymmetry of left-right epaxial muscle (EM) marked by dotted lines) at 307 days of age. Scale bars, 200 microns. EM- epaxial muscle. (H) Representative images from zebrafish embryos injected with GFP2A-PAX3 and GFP2A-PAX3FOXO1 that are fixed at 24 hr post-injection and then TUNEL performed (rhodamine). Embryos were counter-stained for GFP to indicate transgene expression. (I) Quantification of TUNEL-positive pixels normalized to GFP positive pixels, indicated a higher proportion of PAX3-FOXO1 cells were undergoing apoptosis. Error bars represent SD, n = 6–8 embryos per group, * indicates p<0.05, two-tailed Student’s t-test.

Figure 2—figure supplement 1. The tp53^M214K mutation modifies the CMV-PAX3FOXO1 phenotype in developing zebrafish.

(A) Schematic of the experimental strategy to assess the impact of a tp53 mutation on survival, cell tolerance, and apoptosis of mosaic CMV restricted GFP, GFP-PAX3, or 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. All constructs were injected in equimolar amounts relative to 25 ng/µL of CMV-GFP2A-PAX3FOXO1. (C) Quantification of the number of GFP-positive pixels for each embryo imaged at 28 hr post fertilization using the same settings. 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 injected experimental groups. Embryos were fixed at 28 hr post-injection, TUNEL (rhodamine) performed, and then embryos were counter-stained for GFP to denote transgene expression.

Figure 2—figure supplement 2. A model for tp53^M214K mediation of CMV-PAX3FOXO1 RMS tumorigenesis.

GFP-tagged PAX3-FOXO1 was injected at the same concentration into developing wildtype or tp53^M214K homozygous mutant zebrafish. The number of GFP positive and TUNEL-positive cells was assessed at 24 hr post-fertilization with striking differences. In wildtype zebrafish, there is a reduction in the number of GFP-PAX3FOXO1 + cells that is coupled with a significant increase in the number of cells undergoing apoptosis. This embryonic elimination of PAX3-FOXO1 + cells may explain the lack of tumors that develop in CMV-PAX3FOXO1 injected wildtype zebrafish. However, in tp53^M214K mutant zebrafish, there is an increase in the number of GFP-PAX3FOXO1 + cells that is coupled with a decrease in the number of cells undergoing apoptosis. This suggests that inhibition of apoptosis via a tp53-dependent mechanism is allowing for the persistence of embryonic PAX3-FOXO1 + cells and ultimately RMS tumorigenesis in this model.