Stellate Genes Research Articles

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.

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 β-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.

Comparative characteristics of laryngeal-resident mesenchymal stromal cell populations isolated from distinct sites in the rat larynx

BackgroundAlthough tissue-resident mesenchymal stromal cells (MSCs) in the larynx have been described, their distinct characteristics and roles have not been thoroughly explored. Therefore, we investigated stem cell characteristics and regenerative potentials of single clonal populations isolated from rat epiglottic mucosa (EM), lamina propria (LP), and macula flava (MF) to determine whether they comprised laryngeal tissue-resident stem cells.MethodsSingle clonal laryngeal cells were isolated following microdissection of the EM, LP, and MF from the rat larynx. Several clonal populations from the three laryngeal subsites were selected and expanded in vitro. We compared the stem cell characteristics of self-renewal and differentiation potential, as well as the cell surface phenotypes and gene expression profiles, of laryngeal MSC-like cells to that of bone marrow MSCs (BM-MSCs). We also investigated the regenerative potential of the laryngeal cells in a radiation-induced laryngeal injury animal model.ResultsSelf-renewing, clonal cell populations were obtained from rat EM, LP, and MF. EM-derived and LP-derived clonal cells had fibroblast-like features, while MF-resident clonal cells had stellate cell morphology and lipid droplets containing vitamin A. All laryngeal clonal cell populations had MSC-like cell surface marker expression (CD29, CD44, CD73, and CD90) and the potential to differentiate into bone and cartilage cell lineages; EM-derived and MF-derived cells, but not LP-derived cells, were also able to differentiate into adipocytes. Clonal cells isolated from the laryngeal subsites exhibited differential extracellular matrix-related gene expression. We found that the mesenchymal and stellate cell-related genes desmin and nestin were enriched in laryngeal MSC-like cells relative to BM-MSCs (P < 0.001). Growth differentiation factor 3 (GDF3) and glial fibrillary acidic protein (GFAP) transcript and protein levels were higher in MF-derived cells than in other laryngeal populations (P < 0.001). At 4 weeks after transplantation, laryngeal MF-derived and EM-derived cells contributed to laryngeal epithelial and/or glandular regeneration in response to radiation injury.ConclusionsThese results suggest that cell populations with MSC characteristics reside in the EM, LP, and MF of the larynx. Laryngeal MSC-like cells contribute to regeneration of the larynx following injury; further investigation is needed to clarify the differential roles of the populations in laryngeal tissue regeneration, as well as the clinical implications for the treatment of laryngeal disease.

New flower shapes in Linum grandiflorum Desf. and their inheritance

The inbred lines of Linum grandiflorum that differ in the shape of a flower, F1 and F2 population derived from the crosses involving the inbreds were used to study flower shape inheritance pattern. Some lines had shortened petals and in the shape of a flower resembled wild carnations. Another line was distinguished by the strongly involute edges of the petals which gave the flower a stellate shape. F1 plants from the cross between carnation-flowered and stellate-flowered plants with wild type lines (non-shortened petals, non-stellate flower) and also among themselves had wild type of flower shape. The F2 showed a 15: 1 ratio of normal-petalled (wild type) and shortened-petalled plants indicating the control of the carnation shape of flower (“cnf” = carnation flower) by two uniquely acting genes. Stellate shape of flower (“st” = stellate flower) in crosses with nonstellate lines was inherited as monogenic recessive trait showing in F2 a 3: 1 ratio of non-stellate and stellate plants. Stellate and carnation shapes of a flower were determined by different genetic systems and inherited independently. In the case where the parents of the F2 hybrids differed not only in the shape of the flower, but also in its color, the independent inheritance of the genes determining the carnation type of the flower and the three-locus system responsible for its coloring was established. Independent inheritance of the stellate flower gene and Si-locus, the recessive allele of which lightens the color of the petals and the spot in the center of the flower was also shown.

RNA helicase Spn-E is required to maintain Aub and AGO3 protein levels for piRNA silencing in the germline of Drosophila

Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.

Stellate cells and liver parenchyma gene transcription changes after stem cells therapy in experimental liver fibrosis and cirrhosis

Stellate cells and liver parenchyma gene transcription changes after stem cells therapy in experimental liver fibrosis and cirrhosis

Mapping of cis-regulatory sites in the promoter of testis-specific Stellate genes of Drosophila melanogaster

Tandem Stellate genes organized into two clusters in heterochromatin and euchromatin of the X-chromosome are part of the Ste-Su(Ste) genetic system required for maintenance of male fertility and reproduction of Drosophila melanogaster. Stellate genes encode a regulatory subunit of protein kinase CK2 and are the main targets of germline-specific piRNA-silencing; their derepression leads to appearance of protein crystals in spermatocytes, meiotic disturbances, and male sterility. A short promoter region of 134 bp appears to be sufficient for testis-specific transcription of Stellate, and it contains three closely located cis-regulatory elements called E-boxes. By using reporter analysis, we confirmed a strong functionality of the E-boxes in the Stellate promoter for in vivo transcription. Using selective mutagenesis, we have shown that the presence of the central E-box 2 is preferable to maintain a high-level testis-specific transcription of the reporter gene under the Stellate promoter. The Stellate promoter provides transcription even in heterochromatin, and corresponding mRNAs are translated with the generation of full-size protein products in case of disturbances in the piRNA-silencing process. We have also shown for the first time that the activity of the Stellate promoter is determined by chromatin context of the X-chromosome in male germinal cells, and it increases at about twofold when relocating in autosomes.

Expansion and Evolution of the X-Linked Testis Specific Multigene Families in the melanogaster Species Subgroup

The testis specific X-linked genes whose evolution is traced here in the melanogaster species subgroup are thought to undergo fast rate of diversification. The CK2ßtes and NACβtes gene families encode the diverged regulatory β-subunits of protein kinase CK2 and the homologs of β-subunit of nascent peptide associated complex, respectively. We annotated the CK2βtes-like genes related to CK2ßtes family in the D. simulans and D. sechellia genomes. The ancestor CK2βtes-like genes preserved in D. simulans and D. sechellia are considered to be intermediates in the emergence of the D. melanogaster specific Stellate genes related to the CK2ßtes family. The CK2ßtes-like genes are more similar to the unique autosomal CK2ßtes gene than to Stellates, taking into account their peculiarities of polymorphism. The formation of a variant the CK2ßtes gene Stellate in D. melanogaster as a result of illegitimate recombination between a NACßtes promoter and a distinct polymorphic variant of CK2ßtes-like ancestor copy was traced. We found a close nonrandom proximity between the dispersed defective copies of DINE-1 transposons, the members of Helitron family, and the CK2βtes and NACβtes genes, suggesting an involvement of DINE-1 elements in duplication and amplification of these genes.

Promoter contribution to the testis-specific expression of Stellate gene family in Drosophila melanogaster

Testis-specific tandemly repeated Stellate genes are part of the Ste–Su(Ste) genetic system required for male fertility in Drosophila melanogaster. Stellate genes encode a functional homolog of the β-subunit of protein kinase CK2. Derepression of Stellate results in their over-expression, meiotic disturbances and male sterility. Stellate genes are represented by clustered copies in the X chromosome and carry promoters shared with another X-chromosome cluster, βNACtes genes, encoding putative β-subunits of the nascent polypeptide-associated complex. Using Electrophoretic Mobility Shift Assay, we revealed in the Stellate promoter three cis-acting elements, E-boxes, the loss of which greatly diminished the reporter gene expression in Drosophila testes. We identified that these E-boxes were recognized by helix–loop–helix protein, dUSF (Drosophila ortholog of mammalian USF) in testis nuclear extract. All three E-boxes were preserved in the promoters of both euchromatic and heterochromatic Stellate clusters. Two analogous E-boxes were detected in the promoters of 5′-copies of the duplicated βNACtes gene pairs, whereas the 3′-copies lacked these sites but possessed a new binding site for a testis protein distinct from dUSF. Here we characterized a new type of testis-specific core promoter and identified dUSF as its interacting transcription factor.

A novel organelle, the piNG-body, in the nuage ofDrosophilamale germ cells is associated with piRNA-mediated gene silencing

Proteins of the PIWI subfamily Aub and AGO3 associated with the germline-specific perinuclear granules (nuage) are involved in the silencing of retrotransposons and other selfish repetitive elements in the Drosophila genome. PIWI proteins and their 25- to 30-nt PIWI-interacting RNA (piRNAs) are considered as key participants of the piRNA pathway. Using immunostaining, we found a large, nuage-associated organelle in the testes, the piNG-body (piRNA nuage giant body), which was significantly more massive than an ordinary nuage granule. This body contains known ovarian nuage proteins, including Vasa, Aub, AGO3, Tud, Spn-E, Bel, Squ, and Cuff, as well as AGO1, the key component of the microRNA pathway. piNG-bodies emerge at the primary spermatocyte stage of spermatogenesis during the period of active transcription. Aub, Vasa, and Tud are located at the periphery of the piNG-body, whereas AGO3 is found in its core. Mutational analysis revealed that Vasa, Aub, and AGO3 were crucial for both the maintenance of the piNG-body structure and the silencing of selfish Stellate repeats. The piNG-body destruction caused by csul mutations that abolish specific posttranslational symmetrical arginine methylation of PIWI proteins is accompanied by strong derepression of Stellate genes known to be silenced via the piRNA pathway.

Genetically Derepressed Nucleoplasmic Stellate Protein in Spermatocytes of D. melanogaster Interacts with the Catalytic Subunit of Protein Kinase 2 and Carries Histone-Like Lysine-Methylated Mark

Summary The X-chromosome-linked clusters of the tandemly repeated testis-specific Stellate genes of Drosophila melanogaster, encoding proteins homologous to the regulatory β-subunit of the protein kinase casein kinase 2 (CK2), are repressed in wild-type males. Derepression of Stellate genes in the absence of the Y chromosome or Y-linked crystal locus ( crystal line) causes accumulation of abundant protein crystals in testes and different meiotic abnormalities, which lead to partial or complete male sterility. To understand the cause of abnormalities in chromosome behavior owing to Stellate overexpression, we studied subcellular localization of Stellate proteins by biochemical fractionation and immunostaining of whole testes. We showed that, apart from the known accumulation of Stellate in crystalline form, soluble Stellate was located exclusively in the nucleoplasm, whereas Stellate crystals were located mainly in the cytoplasm. Coimmunoprecipitation experiments revealed that the α-subunit of the protein kinase CK2 (CK2α) was associated with soluble Stellate. Interaction between soluble Stellate and CK2α in the nucleus could lead to modulations in the phosphorylation of nuclear targets of CK2 and abnormalities in the meiotic segregation of chromosomes. We also observed that Stellate underwent lysine methylation and mimicked trimethyl-H3K9 epigenetic modification of histone H3 tail.

Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster

Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA–Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of β-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases β-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation.

Gene silencing mechanisms mediated by Aubergine–piRNA complexes in Drosophila male gonad

Genetic studies have shown that Aubergine (Aub), one of the Piwi subfamily of Argonautes in Drosophila, is essential for germ cell formation and maintaining fertility. aub mutations lead to the accumulation of retrotransposons in ovaries and testes, and Stellate transcripts in testes. Aub in ovaries associates with a variety of Piwi-interacting RNAs (piRNAs) derived from repetitive intergenic elements including retrotransposons. Here we found that Aub in testes also associates with various kinds of piRNAs. Although in ovaries Aub-associated piRNA populations are quite diverse, piRNAs with Aub in testes show a strong bias. The most abundant piRNAs were those corresponding to antisense transcripts of Suppressor of Stellate [Su(Ste)] genes known to be involved in Stellate gene silencing. The second most abundant class was made up of those from chromosome X and showed strong complementarity to vasa transcripts. Immunopurified Aub-piRNA complexes from testes displayed activity in cleaving target RNA containing sequences complementary to Stellate and vasa transcripts. These results provide the first biochemical insights into gene silencing mechanisms mediated by Aub and piRNAs in fly testes.

An Alien Promoter Capture as a Primary Step of the Evolution of Testes-Expressed Repeats in the Drosophila melanogaster Genome

Fertility of Drosophila melanogaster males is impaired due to the disruption of the silencing of the X-linked, testis-expressed, repeated Stellate (Ste) genes. Ste silencing is mediated by symmetric transcription of the paralogous Y-linked repeats and exerted by an RNA interference (RNAi) mechanism. Here we present a scenario for the origin of the Ste genes and their suppressors. The primary intermediate of their evolution emerged as a result of the acquisition of a preformed alien, testis-specific promoter. This intermediate is identified as a chimeric gene containing coding region of an autosomal gene for testis-specific protein kinase CK2. The 5' region of the chimera has been acquired from a member of a family of testis-expressed X-linked genes of unknown function. We propose that the evolution and amplification of the novel chimeric gene have led to the overproduction of the regulatory CK2 subunit in testes. The evolution of the Y-linked descendants of the primary intermediate resulted in the RNAi-mediated suppression of excessive expression of the X-linked paralogs. The newly detected "dead family" of cognate repeats on the Y chromosome has contributed to the evolution of Ste and its suppressors via gene conversion. Our results show that RNAi silencing, considered as a defense against viruses and transposable elements, may be involved in the evolution of eukaryotic genomes.

322 α(V)β(3) integrin antagonists block hepatic stellate cell migration and profibrogenic gene expression but aggravate biliary fibrosis

322 α(V)β(3) integrin antagonists block hepatic stellate cell migration and profibrogenic gene expression but aggravate biliary fibrosis

The Heterochromatic ABO Locus May Overlap with the Region Containing Repeats Encompassing Mobile Elements and Stellate Genes on the X Chromosome of Drosophila melanogaster

A deficiency of certain heterochromatic regions (ABO loci) of various chromosomes dramatically distorts the early embryo development in the progeny of females carrying mutation in the abnormal oocyte (abo) gene, which is located in euchromatin of chromosome 2. One ABO locus (X-ABO) is in X-heterochromatin distal to the nucleolus organizer. A cluster of the Stellate repeats is located in the same heterochromatic block. Deletions of various fragments from distal heterochromatin were tested for the effect on expression of the abo mutation. The X-ABO locus was assigned to X-chromosomal heterochromatin segment h26 and may include repeats consisting mostly of mobile elements and defective Stellate copies. A major part of the regular Stellate tandem repeats proved to be distal of the X-ABO locus.

The GATE retrotransposon in Drosophila melanogaster: mobility in heterochromatin and aspects of its expression in germline tissues

A full-length copy of the retrotransposon GATE was identified as an insertion in the tandemly repeated, heterochromatic, Stellate genes, which are expressed in the testis of Drosophila melanogaster. Sequencing of this heterochromatic GATE copy revealed that it is closely related to the BEL retrotransposon, a representative of the recently defined BEL-like group of LTR retrotransposons. This copy contains identical LTRs, indicating that the insertion is a recent event. By contrast, the euchromatic part of the D. melanogaster genome contains only profoundly damaged GATE copies or fragments of the transposon. The preferential localization of GATE sequences in heterochromatin was confirmed for the other species in the melanogaster subgroup. The level of GATE expression is dramatically increased in ovaries, but not in testes, of spn-E(1) homozygous flies. We speculate that spn-E is involved in the silencing of GATE via an RNA interference mechanism.

Does Stellate cause meiotic drive in Drosophila melanogaster?

Drosophila melanogaster males deficient for the crystal (cry) locus of the Y chromosome that carry between 15 and 60 copies of the X-linked Stellate (Ste) gene are semisterile, have elevated levels of nondisjunction, produce distorted sperm genotype ratios (meiotic drive), and evince hyperactive transcription of Ste in the testes. Ste seems to be the active element in this system, and it has been proposed that the ancestral Ste gene was "selfish" and increased in frequency because it caused meiotic drive. This hypothetical evolutionary history is based on the idea that Ste overexpression, and not the lack of cry, causes the meiotic drive of cry(-) males. To test whether this is true, we have constructed a Ste-deleted X chromosome and examined the phenotype of Ste(-)/cry(-) males. If hyperactivity of Ste were necessary for the transmission defects seen in cry(-) males, cry(-) males completely deficient for Ste would be normal. Although it is impossible to construct a completely Ste(-) genotype, we find that Ste(-)/cry(-) males have exactly the same phenotype as Ste(+)/cry(-) males. The deletion of all X chromosome Ste copies not only does not eliminate meiotic drive and nondisjunction, but it also does not even reduce them below the levels produced when the X carries 15 copies of Ste.

Molecular Evolution of Tandemly Repeated Heterochromatic Gene Clusters Involving a Switch of Their Function in the Genome of Drosophila melanogaster

Molecular evolution and divergence of paralogous tandem heterochromatic repeats Stellate and Su(Ste) located on the X and Y chromosomes, respectively, are discussed. These repeats appear to emerge as a result of amplification of a unique autosomal euchromatic gene encoding the regulatory β-subunit of the CK2 protein kinase. The autosomal gene and the clusters of heterochromatic repeats are transcribed in testes. A high level of the Stellate expression leads to partial male sterility and abnormal meiosis. The Stellate expression and its adverse effects are suppressed by homologous Su(Ste) repeats. In genome evolution, the open reading frames (ORF) of Stellate and Su(Ste) putative ancestor were maintained by translational selection. Then the coding Su(Ste) function has been damaged and switched to a new one resulting in antisense Su(Ste) transcription and silencing of theStellategenes. Symmetrical (sense and antisense) Su(Ste) transcription causes the formation of a double-strand RNA, which by interference (selective elimination of the homologous gene expression) suppresses the Stellate genes. The biological significance and the driving forces of evolution of the species-specific balanced interaction of Stellateand Su(Ste) repeats remain mysterious.