Patterning of the respiratory dorsal appendages (DAs) on the Drosophila melanogaster eggshell is tightly regulated by epidermal growth factor receptor (EGFR) signaling. Variation in the DA number is observed among Drosophila species; D. melanogaster has two DAs and D. virilis has four. Diversification in the expression pattern of rhomboid (rho), which activates EGFR signaling in somatic follicle cells, could cause the evolutionary divergence of DA numbers. Here we identified a cis-regulatory element of D. virilis rho. A comparison with D. melanogaster rho enhancer and activity studies in homologous and heterologous species suggested that these rho enhancers did not functionally diverge significantly during the evolution of these species. Experiments using chimeric eggs composed of a D. virilis oocyte and D. melanogaster follicle cells showed the evolution of DA number was not attributable to germline Gurken (Grk) signaling, but to divergence in events downstream of Grk signaling affecting the rho enhancer activity in somatic follicle cells. We found that a transcription factor, Mirror, which activates rho, could be one of these downstream factors. Thus, evolution of the trans-regulatory environment that controls rho expression in somatic follicle cells could be a major contributor to the evolutionary changes in DA number.