Fig. 2. Regulation of SG1 by Fkh is indirect. (A) 972 nucleotides of sequence upstream and spanning the translation start site (green letters) of SG1 were sufficient to direct Fkh-dependent salivary gland expression of the lacZ reporter gene. Only two of the four putative Fkh-binding sites in the 972 nucleotide sequence (b and d, blue sequence and filled blue ovals) were bound by Fkh protein in vitro and competed for binding (Aa, Ab; arrowheads indicate P³²-labelled oligos, the migration of which in the gel has been slowed owing to Fkh binding; the triangles on the top of each gel indicate relative concentrations of cold competitor). Whereas unlabelled wild-type d oligos competed well for binding with labeled wild-type d, the mut d oligo did not compete (compare left and right gels in Aa, arrowheads). Wild-type b oligos competed less well than did wild-type d for binding but better than mut d (compare gel in Ab with gels in Aa, arrowheads). Three consensus bHLH binding sites (CAXXTG) are present in the 972 nucleotide SG1 enhancer (red sequences and red filled circles). (B-D) Wild-type versions of the SG1 972-lacZ construct gave high level ßgal expression in the salivary gland (arrows) and variable expression in other tissues (non-salivary gland staining in B-I corresponds to hemocytes). (E-G) Mutations in the two in vitro Fkh-binding sites (b and d) did not affect the salivary gland expression of the reporter constructs. (H,I) Expression of the SG1 972-lacZ construct was absent in salivary glands (arrows) of fkh mutants, although expression in other tissues (hemocytes) was unaffected (here, embryos were stained in the presence of NiCl, causing the reaction substrate to turn purple/black instead of red/brown, as in the embryos in B-G).