In Drosophila melanogaster, sexual differentiation of the male and female gonads is controlled by the key sex-specific transcription factor Doublesex (DSX). While homologs of DSX are known to control sexual development in virtually all animals, including humans, the downstream mechanism and targets for these proteins remain largely unknown. Previously, our genomic analysis of DSX identified fruitless (fru) as a candidate downstream target of DSX to direct male gonad development. FRU is expressed in key somatic cells of the testis stem cell system (the hub, cyst stem cells and early cyst cells) but is not found in the ovary. It is well known that fru, is regulated by sex-specific alternative splicing to promote male-specific expression. Surprisingly, we found that male-specific FRU expression in the gonad is not caused by TRA/TRA-2 mediated alternative splicing and, instead, DSXM is necessary and sufficient to turn on FRU in the gonad regardless of the chromosomal sex. Our genomic analysis indicates that DSX directly binds to fru P3 and P4 promoters to regulates fru expression at the transcriptional level.
A key step in establishing gonadal sex identity is the formation of sexually-dimorphic gonad stem cell niche. We previously found that dsx is required at 3rd larval instar to actively maintain the male niche identity and inhibit transdifferentiation into a female niche. The temporal expression pattern of FRU correlates with this process and our genetic analysis indicates that fru is required to maintain male niche identity. We also found that fru is required in the adult for both cyst stem cell maintenance and cyst cell differentiation. We propose that fru functions downstream of dsx in the male gonad as a masculinizing factor to maintain the hub identity and cyst stem cell homeostasis.