Effects of binding site mutagenesis on reporter gene expression. Flies with the wild-type cooc310, cooc102, or mib2_Fcenhancer driving nuclear lacZ were crossed to flies with mutated versions of the CRMs driving cytoplasmic GFP and the resulting embryos double-stained for both reporters (lacZ, magenta; GFP, green; overlap, white). Areas of direct overlap are limited due to the nuclear vs. cytoplasmic expression of the two reporters but coincident expression can readily be observed. Embryos are oriented anterior to the left and dorsal to the top, except for panel H, which is a dorsal view. (A-D) Mutagenesis of dTcf, Tin, Pnt, or Twi predicted binding sites in the notum_cooc310 CRM have no discernable effect on CRM activity. (E) Mutation of the Tin site in CRM jhamt_cooc102 causes a quantitative reduction in reporter gene expression but has no effect on expression pattern (compare with Fig. 1E). (F) An intact dTcf site is required for expression mediated by jhamt_cooc102 in the anterior (arrow) but has no effect on the remainder of the reporter gene expression. (G) Mutation of the jhamt_cooc102 Pnt site leads to a near-total loss of reporter gene expression. Arrows indicate a few remaining GFP-positive cells. (H) Putative Pnt binding sites in the mib2_FCenhancer CRM lead to an expansion of reporter gene expression throughout the trunk visceral mesoderm when mutated (arrows) and to additional cells with reporter gene expression in the ventral midline (panel I). Somatic mesoderm cells around the periphery of the pictured embryo express both reporters. (I) Close-up view of the stage 11 ventral midline showing additional cells expressing the mutated mib2_FCenhancer reporter gene (arrows). Arrowheads mark cells which also have expression driven by the wild-type enhancer. (J) In a yan- background (yanXE18), additional cells express the wild-type mib2_FCenhancer reporter gene (arrows). The smaller apparent size of these cells compared to the similar arrow-marked cells in panel I is mainly due to the cytoplasmic vs. nuclear nature of the two reporter genes, although we cannot fully rule out additional ectopic expression using the mutated enhancer. Arrowheads indicate the same wild-type mib2_FCenhancer-expressing cells marked with arrowheads in panel I.