Essential functions of RNA helicase Vasa in maintaining germline stem cells and piRNA-guided Stellate silencing in Drosophila spermatogenesis.

DEAD-box RNA helicase Vasa is required for gonad development and fertility in multiple animals. Vasa is implicated in many crucial aspects of Drosophila oogenesis, including translation regulation, primordial germ cell specification, piRNA silencing of transposable elements, and maintenance of germline stem cells (GSCs). However, data about Vasa functions in Drosophila spermatogenesis remain controversial. Here we showed that loss-of-function vasa mutations led to failures of GSC maintenance in the testes, a severe loss of total germ cell content, and a cessation of male fertility over time. Defects in GSC maintenance in vasa mutant testes were not associated with an increasing frequency of programmed cell death, indicating that a premature loss of GSCs occurred via entering differentiation. We found that Vasa is implicated in the positive regulation of rhino expression both in the testes and ovaries. The introduction of a transgene copy of rhino, encoding a nuclear component of piRNA pathway machinery, in vasa mutant background allowed us to restore premeiotic stages of spermatogenesis, including the maintenance of GSCs and the development of spermatogonia and spermatocytes. However, piRNA-guided repression of Stellate genes in spermatocytes of vasa mutant testes with additional rhino copy was not restored, and male fertility was disrupted. Our study uncovered a novel mechanistic link involving Vasa and Rhino in a regulatory network that mediates GSC maintenance but is dispensable for the perfect biogenesis of Su(Ste) piRNAs in testes. Thus, we have shown that Vasa functions in spermatogenesis are essential at two distinct developmental stages: in GSCs for their maintenance and in spermatocytes for piRNA-mediated silencing of Stellate genes.

Molecular Insights into Female Hybrid Sterility in Interspecific Crosses between Drosophila melanogaster and Drosophila simulans.

Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. Here, we performed an analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDCmel complex in hybrid ovaries was not assembled. However, the activity of the RDCsim complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial AT-chX piRNA targeting of the vasasim allele and a significant zygotic delay in vasamel expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species.

Comparative transcriptional analysis uncovers molecular processes in early and mature somatic cyst cells of Drosophila testes.

The tight interaction between somatic and germline cells is conserved in animal spermatogenesis. The testes of Drosophila melanogaster are the model of choice to identify processes responsible for mature gamete production. However, processes of differentiation and soma-germline interactions occurring in somatic cyst cells are currently understudied. Here we focused on the comparison of transcriptome expression patterns of early and mature somatic cyst cells to find out the developmental changes taking place in them. We employed a FACS-based approach for the isolation of early and mature somatic cyst cells from fly testes, subsequent preparation of RNA-Seq libraries, and analysis of gene differential expression in the sorted cells. We found increased expression of genes involved in cell cycle-related processes in early cyst cells, which is necessary for the proliferation and self-renewal of a crucial population of early cyst cells, cyst stem cells. Genes proposedly required for lamellipodium-like projection organization for proper cyst formation were also detected among the upregulated ones in early cyst cells. Gene Ontology and interactome analyses of upregulated genes in mature cyst cells revealed a striking over-representation of gene categories responsible for metabolic and catabolic cellular processes, as well as genes supporting the energetic state of the cells provided by oxidative phosphorylation that is carried out in mitochondria. Our comparative analyses of differentially expressed genes revealed major peculiarities in early and mature cyst cells and provide novel insight into their regulation, which is important for male fertility.

Drosophila as a Model System for Studying of the Evolution and Functional Specialization of the Y Chromosome.

The Y chromosome is one of the sex chromosomes found in males of animals of different taxa, including insects and mammals. Among all chromosomes, the Y chromosome is characterized by a unique chromatin landscape undergoing dynamic evolutionary change. Being entirely heterochromatic, the Y chromosome as a rule preserves few functional genes, but is enriched in tandem repeats and transposons. Due to difficulties in the assembly of the highly repetitive Y chromosome sequence, deep analyses of Y chromosome evolution, structure, and functions are limited to a few species, one of them being Drosophila melanogaster. Despite Y chromosomes exhibiting high structural divergence between even closely related species, Y-linked genes have evolved convergently and are mainly associated with spermatogenesis-related activities. This indicates that male-specific selection is a dominant force shaping evolution of Y chromosomes across species. This review presents our analysis of current knowledge concerning Y chromosome functions, focusing on recent findings in Drosophila. Here we dissect the experimental and bioinformatics data about the Y chromosome accumulated to date in Drosophila species, providing comparative analysis with mammals, and discussing the relevance of our analysis to a wide range of eukaryotic organisms, including humans.

piRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D. melanogaster male germline.

Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. We found two piRNA clusters that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene.

Functional Significance of Satellite DNAs: Insights From Drosophila.

Since their discovery more than 60 years ago, satellite repeats are still one of the most enigmatic parts of eukaryotic genomes. Being non-coding DNA, satellites were earlier considered to be non-functional "junk," but recently this concept has been extensively revised. Satellite DNA contributes to the essential processes of formation of crucial chromosome structures, heterochromatin establishment, dosage compensation, reproductive isolation, genome stability and development. Genomic abundance of satellites is under stabilizing selection owing of their role in the maintenance of vital regions of the genome - centromeres, pericentromeric regions, and telomeres. Many satellites are transcribed with the generation of long or small non-coding RNAs. Misregulation of their expression is found to lead to various defects in the maintenance of genomic architecture, chromosome segregation and gametogenesis. This review summarizes our current knowledge concerning satellite functions, the mechanisms of regulation and evolution of satellites, focusing on recent findings in Drosophila. We discuss here experimental and bioinformatics data obtained in Drosophila in recent years, suggesting relevance of our analysis to a wide range of eukaryotic organisms.

Stellate Genes and the piRNA Pathway in Speciation and Reproductive Isolation of Drosophila melanogaster.

One of the main conditions of the species splitting from a common precursor lineage is the prevention of a gene flow between diverging populations. The study of Drosophila interspecific hybrids allows to reconstruct the speciation mechanisms and to identify hybrid incompatibility factors that maintain post-zygotic reproductive isolation between closely related species. The regulation, evolution, and maintenance of the testis-specific Ste-Su(Ste) genetic system in Drosophila melanogaster is the subject of investigation worldwide. X-linked tandem testis-specific Stellate genes encode proteins homologous to the regulatory ß-subunit of protein kinase CK2, but they are permanently repressed in wild-type flies by the piRNA pathway via piRNAs originating from the homologous Y-linked Su(Ste) locus. Derepression of Stellate genes caused by Su(Ste) piRNA biogenesis disruption leads to the accumulation of crystalline aggregates in spermatocytes, meiotic defects and male sterility. In this review we summarize current data about the origin, organization, evolution of the Ste-Su(Ste) system, and piRNA-dependent regulation of Stellate expression. The Ste-Su(Ste) system is fixed only in the D. melanogaster genome. According to our hypothesis, the acquisition of the Ste-Su(Ste) system by a part of the ancient fly population appears to be the causative factor of hybrid sterility in crosses of female flies with males that do not carry Y-linked Su(Ste) repeats. To support this scenario, we have directly demonstrated Stellate derepression and the corresponding meiotic disorders in the testes of interspecies hybrids between D. melanogaster and D. mauritiana. This finding embraces our hypothesis about the contribution of the Ste-Su(Ste) system and the piRNA pathway to the emergence of reproductive isolation of D. melanogaster lineage from initial species.

piRNA silencing contributes to interspecies hybrid sterility and reproductive isolation in Drosophila melanogaster.

The piRNA pathway is an adaptive mechanism that maintains genome stability by repression of selfish genomic elements. In the male germline of Drosophila melanogaster repression of Stellate genes by piRNAs generated from Supressor of Stellate (Su(Ste)) locus is required for male fertility, but both Su(Ste) piRNAs and their targets are absent in other Drosophila species. We found that D. melanogaster genome contains multiple X-linked non-coding genomic repeats that have sequence similarity to the protein-coding host gene vasa. In the male germline, these vasa-related AT-chX repeats produce abundant piRNAs that are antisense to vasa; however, vasa mRNA escapes silencing due to imperfect complementarity to AT-chX piRNAs. Unexpectedly, we discovered AT-chX piRNAs target vasa of Drosophila mauritiana in the testes of interspecies hybrids. In the majority of hybrid flies, the testes were strongly reduced in size and germline content. A minority of hybrids maintained wild-type array of premeiotic germ cells in the testes, but in them harmful Stellate genes were derepressed due to the absence of Su(Ste) piRNAs, and meiotic failures were observed. Thus, the piRNA pathway contributes to reproductive isolation between D. melanogaster and closely related species, causing hybrid male sterility via misregulation of two different host protein factors.