Rewired gene interactions during development of serially homologous appendages in male and female Drosophila

Drosophila genitalia and legs are considered to be serially homologous appendages derived from a ventral appendage ‘ground state’ shaped by different Hox inputs. Despite their shared origin, there has been relatively little comparison between the downstream gene regulatory networks and how they may differently pattern and shape these appendages. In the pre-tarsal region of the developing leg and antennae of Drosophila , a combination of the transcription factors C15, Lim1 homeobox 1 (Lim1), and Al (Aristaless) are required for the development of the leg tarsal claws and antennal aristae. However, the roles and interactions of these factors in genital development, and their relationship with other leg-patterning genes, remained unexplored. Here, we investigated the expression and function of C15, Lim1, and Al in the development of male and female terminalia (genitalia and analia). We found that C15 plays distinct roles in the different sexes, repressing male clasper bristle formation while promoting bristle development in the female epiproct. Notably, unlike in the antennal and leg discs, C15, Lim1, and Al are not all co-expressed in any anal or genital structures in either sex, indicating that the interactions among these factors have diverged across serially homologous appendages. Nevertheless, we inferred regulatory interactions between C15 and other factors, including bric a brac 2 and Distal-less, reflecting similarities between leg and genital development consistent with their homology. Finally, we identified a male-specific enhancer of C15 , likely regulated by the Hox gene Abdominal-B ( Abd-B ), which is active in male claspers but not in the female terminalia, legs or antennae. This C15 enhancer modularity may underpin tissue-specific regulatory logic, presumably programmed by distinct Hox inputs such as Abd-B in the genitalia, and could contribute to the diversification of serially homologous ventral appendages.