Transgenic and knockout analyses of Masculinizer and doublesex illuminated the unique functions of doublesex in germ cell sexual development of the silkworm, Bombyxmori

Abstract

Background Masculinizer (Masc) plays a pivotal role in male sex determination in the silkworm, Bombyx mori. Masc is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. The male isoform of Bmdsx (BmdsxM) induces male differentiation in somatic cells, while females express the female isoform of Bmdsx (BmdsxF), which promotes female differentiation in somatic cells. However, the importance of Bmdsx in sexual differentiation in germ cells remains unclear. In Drosophila melanogaster, mechanisms regulating sexual differentiation differ between germ cells and somatic cells. dsx is not required within female or male germ cells for sexual development. However, it remains unclear whether this is also the case in other insect species. Results In this study, we performed genetic analyses using a transgenic line that expressed Masc, as well as various Bmdsx knockout lines. Masc-expressing females express both BmdsxF and BmdsxM and have degenerated ovaries combined with testis-like tissues, which produce sperm. We found that Masc-expressing females with a homozygous mutation in BmdsxM showed normal development in ovaries. The formation of testis-like tissues was abolished in these females. In comparison, Masc-expressing females carrying a homozygous mutation in BmdsxF exhibited almost complete male-specific development in gonads and germ cells. These results suggest that BmdsxM can induce male development in germ cells and internal genital organs, while BmdsxF inhibits BmdsxM activity and represses male differentiation. To investigate whether MASC directly controls male-specific splicing of Bmdsx and identify RNAs that form complexes with MASC in testes, we performed RNA immunoprecipitation (RIP) using an anti-MASC antibody. We found that MASC formed a complex with Bmdsx-AS1 lncRNA, which is a testis-specific factor involved in the male-specific splicing of Bmdsx pre-mRNA. Conclusions Combined, our findings suggest that Masc induces male differentiation in gonads and germ cells by interacting with Bmdsx-AS1 lncRNA and enhancing the production of BmdsxM. Unlike Drosophila dsx, BmdsxM was able to induce spermatogenesis in genetically female (ZW) germ cells. To our knowledge, this is the first report indicating the role of dsx in germ cell sexual development differs among insect species.