Sex-determination hierarchy. Sex-biased expression was controlled for by using mutations in sex-determination genes. Relevant aspects of (a) wild-type female, (b) wild-type males, (c) somatic transformation from female to male (sex transformed), (d) somatic transformation from male to female (sex transformed), and (e) germline transformation are outlined. (a) Sex determination occurs in early embryogenesis, before the activation of dosage compensation, which leads to higher levels of expression of transcription factors on the X chromosome in XX;AA than X;AA individuals. These transcription factors activate Sex-lethal (Sxl) in the soma. The Sxl protein regulates the alternative splicing of the transformer (tra) pre-mRNA such that Tra protein is produced only in females. Sxl also inhibits the formation of the MSL dosage compensation complex. Tra protein and non-sex-specifically expressed Transformer2 (Tra2) protein control the alternative splicing of the doublesex (dsx) pre-mRNA. The dsx mRNAs resulting from Tra- and Tra2-mediated splicing encode a female-specific DsxF transcription factor. Sex determination in the germline is poorly understood and controversial, but a female somatic environment and an independent reading of the XX;AA karyotype in germ cells increases expression of positively acting Ovo transcription factors and their direct target, ovarian tumor (otu). The otu locus is required for Sxl activity in the germline. Note that Sxl does not regulate the MSLs in the germline. The female sex-determination hierarchy results in oogenic differentiation. (b) In X;AA flies Sxl protein is not present. This permits the formation of the MSL dosage-compensation complex. The tra pre-mRNAs are spliced to a non-coding form in the absence of Sxl, and in the absence of Tra protein the dsx pre-mRNA is spliced into a default form encoding a male-specific DsxM transcription factor. The germ cells develop into sperm. (c) XX;AA flies are transformed from females into males using null mutations of tra2 and by using a dsx mutation encoding a pre-mRNA that is constitutively spliced into the male-specific form (dsxswe). Flies bearing dsxswein trans to a deletion produce DsxM protein and no DsxF protein. Similarly, flies null for tra2 produce only DsxM. To remove germline expression from the analysis of somatic X chromosome dosage compensation we took advantage of the fact that XX;AA flies transformed from females into males usually have no germline. These germline-atrophic (having few to no germ cells) XX;AA females transformed into males were compared with X;AA male carcasses (everything but the gonads) or with X;AA males with a genetically ablated germline due to the absence of maternal tud+. (d) X;AA flies are transformed from males into females by expressing female-specific tra cDNA transgenes. The activation of female rather than male sexual differentiation in the X;AA soma results in vast numbers of non-differentiated germ cells, presumably due to sexual incompatibility between the soma and germline. The sexual identity of these cells is ambiguous. (e) Mutations in Sxl (using allelic combinations effecting only in the germline isoforms of Sxl) or otu also result in vast numbers of non-differentiated germ cells. Positive (arrows) and negative (barred lines) genetic or molecular regulation are indicated. Loss-of-function (red) and gain-of-function (green) mutations and phenotypes are indicated.