A male-specific doublesex isoform reveals an evolutionary pathway of sexual development via distinct alternative splicing mechanisms.

The doublesex/mab-3 related transcription factor (Dmrt) genes regulate sexual development in metazoans. Studies of the doublesex (dsx) gene in insects, in particular Drosophila melanogaster, reveal that alternative splicing of dsx generates sex-specific Dsx isoforms underlying sexual differentiation. Such a splicing-based mechanism underlying sex-specific Dmrt function is thought to be evolved from a transcription-based mechanism used in non-insect species, but how such transition occurs during evolution is not known. Here we identified a male-specific dsx transcript (dsx M2 ) through intron retention (IR), in addition to previously identified dsx M and dsx F transcripts through alternative polyadenylation (APA) with mutually exclusive exons. We found that Dsx M2 had similarly masculinizing function as Dsx M . We also found that the IR-based mechanism generating sex-specific dsx transcripts was conserved from flies to cockroaches. Further analysis of these dsx transcripts suggested an evolutionary pathway from sexually monomorphic to sex-specific dsx via the sequential use of IR-based and APA-based alternative splicing.