Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori

Abstract

Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.

The Sex Determination Cascade in the Silkworm

“…In addition, our co-immunoprecipitation experiments suggested that Gtsf1 interacts with SIWI. Such an interaction was also observed in Drosophila [ 28 , 53 , 54 ]. Interestingly, not every component from the piRNA pathway participates in feminization.…”

“…Fem silences a gene unique to Lepidoptera, Masc , which is essential for both male determination and repression of the vital process of dosage compensation of Z-linked genes during embryogenesis ( Figure 1 ) [ 21 , 26 , 33 , 34 , 35 ]. In recent years, sex-determined factors encoded by PSI , Znf-2, Siwi and Gtsf1 have been identified along with preliminary established genetic cascade of the sex determination in the silkworm [ 25 , 27 , 28 , 33 , 36 ].…”

“…However, several key elements of the piRNA pathway in the fly, such as Yb, Rhino, Deadlock and Cutoff, which are crucial for piRNA transcription initiation and piRNA processing, are absent in the silkworm [ 52 ]. We have reported that a conserved component of the piRNA pathway called Gtsf1 is involved in female sex determination in the silkworm [ 28 ]. In Drosophila , Gtsf1 is required for female fertility and interacts with PIWI via its C-terminal end; and it is also essential for piRISCs-induced transposon silencing but not for piRNA biogenesis [ 53 , 54 ].…”

“…In Drosophila , Gtsf1 is required for female fertility and interacts with PIWI via its C-terminal end; and it is also essential for piRISCs-induced transposon silencing but not for piRNA biogenesis [ 53 , 54 ]. In the silkworm Gtsf1 is not only necessary for transposon silencing but also for piRNA biogenesis [ 28 ]. In addition, our co-immunoprecipitation experiments suggested that Gtsf1 interacts with SIWI.…”

“…This implies that the sex determiner is a female factor (F-factor) from the W chromosome [ 23 ]. Studies using high-throughput sequencing and genome editing technologies have identified unique sex determination genes in lepidopterans such as Silkworm-PIWI ( Siwi ), gametocyte-specific factor 1 ( Gtsf1 ), Masculinizer ( Masc ) and P-element somatic inhibitor (PSI ), and have preliminarily established the molecular mechanism of the sex determination cascade in Lepidoptera [ 21 , 24 , 25 , 26 , 27 , 28 ] ( Figure 1 ). Here we summarize the progresses of sex determination studies achieved in recent years in lepidopteran and compare them with the model D. melanogaster .…”