Supporting Information

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Fig. 6. The PAX-FKHR fusion protein. The structures of PAX3/7 (the PAX3 gene is on chromosome 2, the PAX7 gene is on chromosome 1), FKHR, and the PAX3/7-FKHR fusion are shown. The vertical dashed line indicates the fusion point. Because all characterized ARMS translocations have been shown to occur within the same PAX and FKHR introns, the PAX-FKHR fusion protein is identical for each tumor. The PAX paired domain and homeodomain DNA-binding motifs, shown in green, are intact in the PAX-FKHR fusion. The FKHR Forkhead DNA binding domain (black box) is severely truncated in the fusion.

Fig. 7. The PAX3/7 subfamily of PAX is conserved in Drosophila. Shown is an amino acid sequence alignment of human PAX3 and PAX7 and the Drosophila orthologs Gooseberry (Gsb) and Gooseberry-neuro (Gsb Neuro). For PAX 3/7, only the portion of the protein present in the PAX-FKHR fusion is shown. For Gsb/Gsb-neuro, the sequence of the full-length protein is truncated to correspond to the analogous portion of PAX3/7 present in the fusion. The paired DNA binding domain in shown and underlined in orange; the homeodomain is in bright blue; the octapeptide motif is in green. Dm, D. melanogaster; Hs, Homo sapiens.

Fig. 8. A first-instar MHC-Gal4; UAS-PAX7-FKHR, UAS-2xeGFP (MHC>>PAX7-FKHR, 2xeGFP) larva. (a) A dark-field image. (b) A GFP fluorescent image. Signal seen is background/autofluorescence. The typical muscle pattern seen in older MHC-Gal4, UAS-PAX7-FKHR, UAS-GFP larvae is not evident.

Fig. 9. Adult myoblasts in PAX7-FKHR and wild-type larvae. (a-a''') The adult myoblasts are present and properly positioned in PAX7-FKHR animals and do not express GFP. Shown are adult myoblasts from an abdominal hemisegment in a nonwandering third-instar larva. (b-b''') The adult myoblasts in control larvae that contain two copies of both the MHC-Gal driver and UAS-2xeGFP reporter do not express GFP. Shown are adult myoblasts from an abdominal hemisegment of a late (wandering) third-instar larva. The adult myoblasts are stained with D-MEF2 antisera and highlighted by the white arrowheads. (Magnifications: ´63.)

Fig. 10. Wild-type PAX3 also causes muscular phenotypes. (a and a') An MHC-Gal4, UAS-GFP, UAS-PAX3 (MHC>>PAX3) larva. The two hemisegments indicated by the white bar are shown in a'. (b and b') A dark-field (b) and GFP fluorescent (b') image of a PAX3 (MHC-Gal4, UAS-GFP, UAS-PAX3) CNS organ. The arrow in b' highlights GFP-positive tissue. (c-c'') High-power confocal images of a GFP-positive PAX3 larval CNS organ. The white arrow highlights the individual nucleus that corresponds to the GFP-positive cell. (Magnification: ´252, c-c''.)

Fig. 11. Histology of abnormal Ras^V12, PAX7-FKHR muscle. (a) A representative histologic profile of normal, syncytial, cross-striated larval muscle. (b and b') Histology of abnormal MHC-Gal4, UAS-Ras^V12, UAS-PAX7-FKHR muscle. b' shows a close-in view of muscular tissue (noted in b by the arrowheads and rotated in b') apparently composed in part of discrete, mononuclear cells (right portion). Images are taken from histologic sections cut along the long axis of larvae embedded in paraffin with the ventral surface "face down" and stained with hematoxylin and eosin. The black arrows in a and b note the outer cuticular surface. (Magnification: ´63, a and b.)