Fig. 9. Twist/Da heterodimers repress somatic muscle formation in vivo. (A-E) Wild-type embryos; (F-J) embryos expressing Da in a heterozygous twist background; (K-O) embryos expressing Twist-Da linked dimer in a wild-type background. Overexpression is achieved using twist-GAL4. (A,F,K) Mhc expression in stage 16 embryos. Embryos expressing Da or Twist-Da in mesoderm show severely reduced numbers of muscle cells. (B,G,L) Kr protein in stage 12 embryos. Kr expression in muscle precursors is highly reduced in embryos overexpressing Da (arrow). This phenotype is more severe in embryos expressing Twist-Da (L, arrow). (C,H,M) Fas III expression in stage 12 embryos. Fas III mesodermal expression is normal in embryos expressing Da protein (H), however some subtle defects can be observed in embryos expressing Twi-Da linked dimer (M; arrowhead). (D,I,N) Bap expression in stage 11 embryos. Progenitor cells of the visceral mesoderm and fat body show normal levels of Bap. (E,J,O) ß-galactosidase expression in stage 10 embryos. Transgenic flies carrying a 175 bp Mef2-lacZ enhancer (the same enhancer used in transient transfection assays) were stained for ß-galactosidase in the absence of ectopically expressed protein (E), in the presence of Da expression in a twist heterozygous background (J), or in the presence of Twist-Da expression in a wild-type background (O). The 175 bp enhancer was active in wild-type embryos, however, expression of Da or Twist-Da caused reduction of enhancer activity as seen in tissue culture cells. Using other GAL4s (i.e., 24BGAL4; Brand and Perrimon, 1993; Baylies et al., 1995) to drive expression of the tethered Twist-Da heterodimer in a wild-type background or Da in the twist heterozygous background resulted in similar defects.