title: Functional analysis of sex-determination genes by gene silencing with LNA-DNA gapmers in the silkworm, Bombyx mori.
abstract: The sexual fate of B. mori is determined genetically; ZW, female and ZZ, male. Recently, we successfully identified a strong candidate gene at the top of the sex determination cascade in B. mori. This gene was termed Feminizer (Fem) and revealed to be a source of Fem-piRNA. Further, we found that B. mori doublesex (Bmdsx) splicing was markedly altered to produce the male-type isoform when a Fem-piRNA inhibitor was injected into ZW embryos. Moreover, knockdown of Masculinizer (Masc), a Fem-piRNA target gene, altered to produce the female-type isoform of Bmdsx in male embryos. However, it remains unclear as to whether Masc directly regulates the sex-specific expression of Bmdsx. In previous studies, we determined that the male-specific isoform of the Bombyx homolog of IGF-II mRNA-binding protein (Imp(M)) was involved in the male-specific splicing of Bmdsx. In an attempt to clarify the genetic relationship between Fem, Masc, Imp(M), and Bmdsx, knockdown experiments were performed. Knockdown of Fem shifted into male-type Bmdsx, Imp(M) and Masc in female embryos. Knockdown of Masc led to the production of the female-type Bmdsx and a dramatic reduction in Imp(M) expression in male embryos. Knockdown of Imp(M) shifted Bmdsx splice mode from the male-type into the female-type. Our results suggest that: (1) Fem reduces Masc expression, (2) Masc dramatically induces Imp(M) expression, and (3) Imp(M) shifting Bmdsx splice mode from the female-type into the male-type. Based on these findings, we propose a possible genetic cascade regulating sex determination in B. mori.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sakai
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.
authors.forename: Honami
authors.initials: H
authors.lastname: Sakaguchi
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.
authors.forename: Fugaku
authors.initials: F
authors.lastname: Aoki
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan. Electronic address: gakyo@k.u-tokyo.ac.jp.
authors.forename: Masataka G
authors.initials: MG
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D012831:Bombyx / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D020868:Gene Silencing / Q000502:physiology*; D019476:Insect Proteins / Q000235:genetics*; D008297:Male; D059368:RNA Isoforms / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D019849:Sex Determination Processes / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D019476:Insect Proteins; D059368:RNA Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering
keywords: Bmdsx; Bombyx mori; Fem; Imp; Masc; Sex determination