ABSTRACT: The Drosophila genitalia develop from an imaginal disc that is patterned during the larval period and undergoes morphogenesis during the pupal period. Although the genetic hierarchy that controls sexual identity in Drosophila is well characterized, the downstream genes that effect sexually dimorphic development of the genitalia are largely unknown. We used microarrays to profile gene expression in female and male genital imaginal discs at three time points during development: late third-instar larvae (L3), pupae approximately 6 hours after puparium formation (P6) and pupae approximately 20 hours after puparium formation (P20). We identified genes that are sex-differentially expressed in the developing genital disc, including three genes encoding transcription factors that are expressed in the genital disc of one sex but not the other. Through genetic analysis, we showed how the master regulator of sexual identity, Doublesex, limits expression of each of these three genes to one sex. We also determined which genital structures fail to develop properly in the absence of each of the three genes. Drosophila female or male genital discs were dissected at the L3, P6 or P20 time points, total RNA was extracted, and labeled cDNA was produced for hybridization to Affymetrix microarrays. To aid in identifying genital discs, and thereby to avoid loss or contamination of tissue, we used animals expressing GFP in all imaginal discs (esgGAL4.B, UAS-GFP.nls/CyO). To collect discs of specific developmental ages, short-duration sex-nonspecific morphological features were used. L3 discs were dissected from wandering third-instar larvae and sexed by gonad size. For the two pupal time points, each sex-sorted larva was observed until it reached the white prepupa stage (0 hours after puparium formation, APF), which lasts 15-20 minutes. This short duration improves synchronization of the following stages. An air bubble forms mid-dorsally on the puparium at around 4 hours APF, peaks in size at 6-6.5 hours APF and disappears at 9-11 hours APF. We therefore used this morphological feature to collect P6 samples, rather than merely timing 6 hours since the white prepupa. Each animal was observed every 20 minutes from 5 hours APF, until peak bubble size was evident, at which time its genital disc was dissected. We followed a similar protocol for P20. We noted that GFP expression in an oval patch in the eye disc becomes faintly visible at around 20 hours APF. We observed each pupa every 20 minutes from 18 hours APF, until GFP appeared in the eye, at which time its genital disc was dissected. Morphologies of P6 and P20 discs collected this way showed little within-sex variation. For each combination of sex and time point, four biological replicates were performed.
