D. willistoni enhancers drive expression in the male genitalia but not in the sex comb. (A) Map of the sex comb (red), genital (black) and early foreleg (purple) enhancers in D. melanogaster. The D. willistoni reporter fragment (orange) encompasses part of the second exon, the genital enhancer, and the region homologous to the sex comb enhancer in between. The VISTA plot (Frazer et al., 2004) below shows regions of high sequence similarity between the two species, which were used to locate the right boundary of the genital enhancer in D. willistoni. Similarity >70% is highlighted in pink. (B) Expression of the D. willistoni reporter in in pupal male genitalia at 45 h APF. The strongest expression is seen in the aedeagal sheath (white arrow), with weaker expression in the clasper (green arrow), suggesting that the genital enhancer is functionally conserved between D. willistoni and D. melanogaster. (C) The same D. willistoni reporter in the pupal foreleg of D. melanogaster at 24 h APF (green), with Dsx counter-stained in red. The reporter shows ectopic expression in ta2, but has no activity in the sex comb region in distal ta1.