Fig 2: Neural transcription factors with known and novel function in the developing cerebellum are enriched in dynamic cerebellar enhancers.(A) Dot plot displaying significantly enriched (adjusted p-value <1E-11) motifs and the predicted matching transcription factor (TF). Displayed are the results for Early (top) and Late (bottom) active enhancers. TFs with an unknown functional role in cerebellar development are indicated with a red arrow. Size of the dots indicate the negative log10 adjusted p-value for a given motif and the color scale displays the z-score normalized expression throughout the cerebellar developmental time course. (B) Top: Immunofluorescent staining of Pax3 in the mouse cerebellum at E12, E15, P0, and P3. Bottom: Pax3 and Ptf1a immunofluorescent co-staining of the E12 mouse cerebellum. Immunofluorescent co-staining of Pax3 and Pax2 in the mouse cerebellum at E15 and P0. Labels: CP: Cerebellar parenchyma, EGL: External granular layer, NGL: Nascent granular layer, RL: Rhombic lip, VZ: Ventricular zone, Scalebars = 100 µm.

Fig 3: eYFP activity and Pax3:Foxo1a expression is cell cycle specific.(A) Diagrammatic representation of the conditional Pax3:Foxo1a knock-in allele by which eYFP is expressed as a second cistron on the same mRNA as Pax3:Foxo1a at the native Pax3 promoter. (B) Heterogeneity of eYFP expression in a murine aRMS tumor by immunofluorescence. (C) eYFP fluorescence of eYFP sorted cells overtime as measured by FACS. Grey: C2C12 (negative control), blue: no sorted cells, green: eYFP activity high cells, red: eYFP activity low cells. (D) Mean of relative eYFP activity in panel 1C measured by FACS. (E) Western blot analysis using eYFP sorted cells. Plotted are relative protein levels of Pax3:Foxo1a/ß-Actin. Mean ± SE were obtained from three independent immunoblottings. Black line shows significant difference (p<0.05). (F) eYFP activity and cell cycle analysis using Hochest33342 staining for mouse primary cell culture U23674. Green shows G0/G1 phase, brown shows S phase, and blue shows G2/M phase. (G) Time-lapse experiment of eYFP activity (select frames over 16 hours). See also corresponding Movie S1.

Fig 4: Treatment-related implications for dynamic oncogene expression in rhabdomyosarcoma in vivo.(A) Kaplan-Meier survival analysis for disease-free survival of mice implanted with pre-irradiated (10Gy) primary murine aRMS tumor cells treated with Pax3:Foxo1a siRNA (siY) or control siRNA (siC), n = 5 animals per cohort. The p value for the difference between siY and siC groups receiving radiation was 0.02. (B) Diagrammatic representation of results in (A).

Fig 5: Pax3:Foxo1a is expressed in G2 for mouse and human aRMS.Immunocytochemistry for Pax3 (green), pHH3 (red) and DAPI (blue) or Pax3 (green), pCDC2 (red) and DAPI (blue). Numbers are relative rate of Pax3:Foxo1a high or low cells/pHH3 positive cells and Pax3:Foxo1a high or low cells/CDC2-Y15 high cells. Black line shows significant difference (p<0.05).