Munetaka Kawamoto

A single female-specific piRNA is the primary determiner of sex in the silkworm

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

The Endosymbiotic Bacterium Wolbachia Selectively Kills Male Hosts by Targeting the Masculinizing Gene

Pathogens are known to manipulate the reproduction and development of their hosts for their own benefit. Wolbachia is an endosymbiotic bacterium that infects a wide range of insect species. Wolbachia is known as an example of a parasite that manipulates the sex of its hosts progeny. Infection of Ostrinia moths by Wolbachia causes the production of all-female progeny, however, the mechanism of how Wolbachia accomplishes this male-specific killing is unknown. Here we show for the first time that Wolbachia targets the host masculinizing gene of Ostrinia to accomplish male-killing. We found that Wolbachia-infected O. furnacalis embryos do not express the male-specific splice variant of doublesex, a gene which acts at the downstream end of the sex differentiation cascade, throughout embryonic development. Transcriptome analysis revealed that Wolbachia infection markedly reduces the mRNA level of Masc, a gene that encodes a protein required for both masculinization and dosage compensation in the silkworm Bombyx mori. Detailed bioinformatic analysis also elucidated that dosage compensation of Z-linked genes fails in Wolbachia-infected O. furnacalis embryos, a phenomenon that is extremely similar to that observed in Masc mRNA-depleted male embryos of B. mori. Finally, injection of in vitro transcribed Masc cRNA into Wolbachia-infected embryos rescued male progeny. Our results show that Wolbachia-induced male-killing is caused by a failure of dosage compensation via repression of the host masculinizing gene. Our study also shows a novel strategy by which a pathogen hijacks the host sex determination cascade.

Albino (al) is a tetrahydrobiopterin (BH4)-deficient mutant of the silkworm Bombyx mori

Albino (al) is a lethal mutant of Bombyx mori that exhibits a colourless cuticle after the first ecdysis and dies without feeding on mulberry. Previous studies have indicated that sclerotisation was insufficient because of defective phenylalanine and tyrosine metabolism in albino larvae. However, the genetic mechanism underlying the albino phenotype has not been determined. Dopamine plays a central role in insect cuticle colouration and sclerotisation. The pathway for dopamine biosynthesis from phenylalanine involves phenylalanine hydroxylase (PAH; EC 1.14.16.1) and tyrosine hydroxylase (TH; EC 1.14.16.2). Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases, including PAH and TH. Thus, BH4 is indispensable for cuticle colouration and sclerotisation. Here we report on identifying mutations in the gene that encodes for the Bombyx homolog of 6-pyruvoyl-tetrahydropterin synthase (PTS) which is involved in the biosynthesis of BH4, in 2 strains with different al alleles. In strain a60 (al), a transposable element was inserted in exon 2 of BmPTS. In strain a61 (al²), an 11-bp deletion was identified in the exon 2 region of BmPTS. After oral administration of BH4 to the al² larvae, the survival rate was effectively increased and the larval integument was pigmented. These results indicated that BmPTS was responsible for the albino mutants of B. mori. We conclude that (i) a mutation in BmPTS leads to an insufficient supply of BH4 and results in defective dopamine biosynthesis and (ii) lack of dopamine results in cuticle colouration and sclerotisation failure. Lemon (lem) is a BH4-deficient mutant. It has been reported that de novo synthesis of zygotic BH4 was indispensable for viability of the embryo in eggs laid by lem (lem/lem¹) females. We found that lem/lem, al²/al² larvae produced by lem (lem/lem) females were viable during the first instar stage, suggesting that al²/al² embryo could synthesis BH4 by using maternally transmitted BmPTS.

Identification and functional analysis of a Masculinizer orthologue in Trilocha varians (Lepidoptera: Bombycidae)

We recently showed that the Masculinizer gene (Masc) plays a primary role in sex determination in the lepidopteran model insect Bombyx mori. However, it remains unknown whether this Masc protein‐dependent sex determination system is conserved amongst lepidopteran insects or within the family Bombycidae. Here we cloned and characterized a Masc homologue (TvMasc) in Trilocha varians (Lepidoptera: Bombycidae), a species closely related to B. mori. To elucidate the role of TvMasc in the sex determination cascade of T. varians, TvMasc expression was knocked down in early embryos by the injection of small interfering RNAs (siRNAs) that targeted TvMasc mRNAs. Both female‐ and male‐type splice variants of Tvdsx, a doublesex (dsx) homologue in T. varians were observed in control siRNA‐injected embryos. By contrast, only female‐type splice variants were observed in TvMasc siRNA‐injected embryos. These results indicate that the TvMasc protein directly or indirectly regulates the splicing patterns of Tvdsx. Furthermore, we found that male‐type splice variants of B. mori dsx (Bmdsx) were produced in TvMasc‐overexpressing BmN4 cells. The mRNA level of B. mori Imp, a gene whose product induces male‐specific Bmdsx splicing also increased. These results suggest that Masc genes play similar roles in the sex‐determination cascade in Bombycidae.

Sexually biased transcripts at early embryonic stages of the silkworm depend on the sex chromosome constitution.

In the silkworm, Bombyx mori, females are heterogametic (WZ) whereas males have two Z chromosomes. Femaleness of B. mori is determined by the presence of the W chromosome, suggesting that there is a dominant feminizing gene on this chromosome. Recently, by transcriptome analysis of B. mori embryos, we discovered that a single W-chromosome-derived PIWI-interacting RNA (piRNA) is the long-sought primary determinant of femaleness in B. mori. However, sexual bias in the transcriptome of B. mori early embryos has not yet been well characterized. Using deep sequencing data from molecularly sexed RNA of B. mori embryos, we identified and characterized 157 transcripts that are statistically differentially expressed between male and female early embryos. Most of the female-biased transcripts were transposons or repeat sequences that are produced presumably from the W chromosome. Bioinformatic analysis revealed that these repetitive sequences are piRNA precursors. In contrast, male-biased genes were frequently transcribed from the Z chromosome, suggesting that dosage compensation in Z-linked genes does not occur or is incomplete at early embryonic stages. Our analysis has drawn a picture of a global landscape of sexually biased transcriptome during early B. mori embyogenesis and has suggested for the first time that most sexually biased embryonic transcripts depend on sex chromosomes.

Guardian small RNAs and sex determination

The W chromosome of the silkworm Bombyx mori has been known to determine femaleness for more than 80 years. However, the feminizing gene has not been molecularly identified, because the B. mori W chromosome is almost fully occupied by a large number of transposable elements. The W chromosome-derived feminizing factor of B. mori was recently shown to be a female-specific PIWI-interacting RNA (piRNA). piRNAs are small RNAs that potentially repress invading “non-self” elements (e.g., transposons and virus-like elements) by associating with PIWI proteins. Our results revealed that female-specific piRNA precursors, which we named Fem, are transcribed from the sex-determining region of the W chromosome at the early embryonic stage and are processed into a single mature piRNA (Fem piRNA). Fem piRNA forms a complex with Siwi (silkworm Piwi), which cleaves a protein-coding mRNA transcribed from the Z chromosome. RNA interference of this Z-linked gene, which we named Masc, revealed that this gene encodes a protein required for masculinization and dosage compensation. Fem and Masc both participate in the ping-pong cycle of the piRNA amplification loop by associating with the 2 B. mori PIWI proteins Siwi and BmAgo3 (silkworm Ago3), respectively, indicating that the piRNA-mediated interaction between the 2 sex chromosomes is the primary signal for the B. mori sex determination cascade. Fem is a non-transposable repetitive sequence on the W chromosome, whereas Masc is a single-copy protein-coding gene. It is of great interest how the piRNA system recognizes “self ”Masc mRNA as “non-self” RNA.

Silkworm HP1a transcriptionally enhances highly expressed euchromatic genes via association with their transcription start sites

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species and is crucial for heterochromatin establishment and maintenance. The silkworm, Bombyx mori, encodes two HP1 proteins, BmHP1a and BmHP1b. In order to investigate the role of BmHP1a in transcriptional regulation, we performed genome-wide analyses of the transcriptome, transcription start sites (TSSs), chromatin modification states and BmHP1a-binding sites of the silkworm ovary-derived BmN4 cell line. We identified a number of BmHP1a-binding loci throughout the silkworm genome and found that these loci included TSSs and frequently co-occurred with neighboring euchromatic histone modifications. In addition, we observed that genes with BmHP1a-associated TSSs were relatively highly expressed in BmN4 cells. RNA interference-mediated BmHP1a depletion resulted in the transcriptional repression of highly expressed genes with BmHP1a-associated TSSs, whereas genes not coupled with BmHP1a-binding regions were less affected by the treatment. These results demonstrate that BmHP1a binds near TSSs of highly expressed euchromatic genes and positively regulates their expression. Our study revealed a novel mode of transcriptional regulation mediated by HP1 proteins.

Characterization of a novel chromodomain-containing gene from the silkworm, Bombyx mori

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species, and is crucial in the establishment and maintenance of heterochromatin. HP1 proteins have two distinct functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), which are required for the selective binding of HP1 proteins to modified histones. During our screen for HP1-like proteins in the Bombyx mori genome, we found a novel silkworm gene, Bombyx mori chromodomain protein 1 (BmCdp1), encoding a putative chromobox protein with only two CDs. The BmCdp1 family proteins are closely related to the HP1 proteins, and most of them belong to insect lineages. qRT-PCR analysis indicated that BmCdp1 mRNA was most abundantly expressed in early embryos, and relatively higher expression was observed in larval testes, hemocytes, and pupal ovaries. Western blot and immunostaining experiments showed that BmCdp1 was localized mainly in the nucleus of BmN4 cells. We searched BmCdp1-bound loci in the Bombyx genome by ChIP-seq analysis using Flag-tagged BmCdp1-expressing BmN4 cells. Combined with ChIP-qPCR experiments, we identified two reliable BmCdp1-bound loci in the genome. siRNA-mediated knockdown of BmCdp1 in BmN4 cells and early embryos did not affect the expression of the gene located close to the BmCdp1-bound locus.

Unique sex determination system in the silkworm, Bombyx mori : current status and beyond

The silkworm Bombyx mori has been used for silk production for over 5,000 years. In addition to its contribution to sericulture, B. mori has played an important role in the field of genetics. Classical genetic studies revealed that a gene(s) with a strong feminizing activity is located on the W chromosome, but this W-linked feminizing gene, called Feminizer (Fem), had not been cloned despite more than 80 years of study. In 2014, we discovered that Fem is a precursor of a single W chromosome-derived PIWI-interacting RNA (piRNA). Fem-derived piRNA binds to PIWI protein, and this complex then cleaves the mRNA of the Z-linked Masculinizer (Masc) gene, which encodes a protein required for both masculinization and dosage compensation. These findings showed that the piRNA-mediated interaction between the two sex chromosomes is the primary signal for the sex determination cascade in B. mori. In this review, we summarize the history, current status, and perspective of studies on sex determination and related topics in B. mori.

Transcriptome profiling reveals infection strategy of an insect maculavirus

Abstract Bombyx mori macula-like virus (BmMLV) is a positive, single-stranded insect RNA virus that is closely related to plant maculaviruses. BmMLV is currently characterized as an unclassified maculavirus. BmMLV accumulates at extremely high levels in cell lines derived from the silkworm, Bombyx mori, but it does not lead to lethality and establishes persistent infections. It is unknown how this insect maculavirus replicates and establishes persistent infections in insect cells. Here, we showed that BmMLV p15, which is located on a subgenomic fragment and is not found in plant maculaviruses, is highly expressed in BmMLV-infected silkworm cells and that p15 protein is required to establish BmMLV infections in silkworm cells. We also showed that two distinct small RNA-mediated pathways maintain BmMLV levels in BmMLV-infected silkworm cells, thereby allowing the virus to establish persistent infection. Virus-derived siRNAs and piRNAs were both produced as the infection progressed. Knockdown experiments demonstrated that the exogenous RNAi pathway alone or RNAi and piRNA pathways function cooperatively to silence BmMLV RNA and that both pathways are important for normal growth of BmMLV-infected silkworm cells. On the basis of our study, we propose a mechanism of how a plant virus-like insect virus can establish persistent infections in insect cells.