A double negative post-transcriptional regulatory circuit mediates the virgin behavioral state

The survival and reproductive success of animals depends on the ability to harmonize their external behaviors with their internal states. For example, females conduct numerous social programs that are distinctive to virgins, compared to post-mated and/or pregnant individuals. In Drosophila, the fact that this post-mating switch is initiated by seminal factors implies that the default state is virgin. However, we recently showed that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgin females to execute mated behaviors. To elucidate new components of this post-transcriptional regulatory circuit, we used genomic analysis of mir-iab-4/8 deletion and hth-miRNA binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. While Dsx has mostly been studied during sex-specific differentiation, its activities in neurons are little known. We find that accumulation of Dsx in the CNS is highly complementary to Hth, and downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose-sensitive to developmental dsx function. Strikingly, depletion of Dsx in SAG-1 cells, a highly restricted set of abdominal neurons, abrogates female virgin conducts in favor of mated behavioral programs. Thus, a double negative post-transcriptional pathway in the VNC (miR-iab-4/8 -| Hth -| Dsx) specifies the virgin behavioral state.