Morphogenesis of the Balbiani body in developing oocytes of an orthopteran, Metrioptera brachyptera, and multiplication of female germline mitochondria.

Balbiani body (Bb) is a female germline specific organelle complex. Although the morphology and morphogenesis of the Bb have been analyzed in numerous vertebrate and invertebrate species, the role and ultimate fate of this organelle assemblage are still under debate. As a result, various functions have been attributed to the Bb in given animal lineages or even species. Our analyses showed that in the bush cricket, Metrioptera brachyptera, the Bb is an elaborate and highly dynamic structure positioned at one side of the oocyte nucleus. It forms in early previtellogenic oocytes and consists of two compartments: perinuclear and cytoplasmic. In the cytoplasmic compartment, characteristic complexes of nuage and polymorphous mitochondria are present. Computer-aided 3D reconstructions revealed that mitochondria clustered around neighboring nuage accumulations remain in a physical contact and form an extensive, though dispersed network. As oogenesis progresses, nuage/mitochondria complexes are partitioned into progressively smaller entities that become separated from each other. Concurrently, the mitochondrial network splits into small individual mitochondria populating the whole ooplasm. Immunohistochemical analysis showed that the latter process involves dynamin-related protein 1 (Drp1). Collectively, our findings suggest that in basal insect species, the Bb might be responsible for the selection as well as multiplication of the oocyte mitochondria.

The vertebrate Balbiani body, germ plasm, and oocyte polarity.

The fate of future generations depends on a high-quality germ line. For a female to successfully produce offspring, her oocytes must be successfully specified and their contents meticulously organized. Germ cells are specified by two general mechanisms: inductive and inherited. In the inductive mechanism, the primordial germ cells (PGCs) are induced by signals from the surrounding cells. In the inherited mechanism, PGCs are specified by passing localized germ plasm material from the oocyte to the future germ cells. The Balbiani body, a conserved oocyte aggregate, facilitates the organization of the oocyte into a polarized cell with discrete cytoplasmic domains, including localizing the germ plasm. In the mouse, the Balbiani body is implicated in oocyte survival, while in frogs and zebrafish the Balbiani body carries specific mRNAs to the vegetal pole. These asymmetric mRNAs form the foundation of the functionally polarized oocyte and play important roles in axial patterning and germ plasm formation of the embryo.

Meiosis, Balbiani body and early asymmetry of Thermobia oocyte.

The meiotic division guarantees maintenance of a genetic diversity; it consists of several stages, with prophase I being the longest and the most complex. We decided to follow the course of initial stages of meiotic division in ovaries of Thermobia domestica using modified techniques of squash preparations, semithin sections, and electron microscopy. We show that germaria contain numerous germline cells that can be classified into three categories: cystoblasts, meiotic oocytes, and growing previtellogenic oocytes. The cystoblasts are located most apically. The meiotic oocytes occupy the middle part of the germarium, and the previtellogenic oocytes can be found in the most basal part, near the vitellarium. Analyses of the semithin sections and squash preparations show that post leptotene meiotic chromosomes gather in one region of the nucleoplasm where they form the so-called bouquet. The telomeres of the bouquet chromosomes are attached to a relatively small area (segment) of the nuclear envelope. Next to this envelope segment, the nucleolar organizers are also located. We show that in concert to sequential changes inside the oocyte nuclei, rearrangement of organelles within the ooplasm (oocyte cytoplasm) takes place. This leads to the formation of the Balbiani body and consequent asymmetry of the ooplasm. These early nuclear and cytoplasmic asymmetries, however, are transient. During diplotene, the chromosome bouquet disappears, while the Balbiani body gradually disperses throughout the ooplasm. Finally, our observations indicate the presence of lampbrush chromosomes in the nuclei of previtellogenic oocytes. In the close vicinity to lampbrush chromosomes, characteristic spherical nuclear bodies are present.

Exclusion of dysfunctional mitochondria from Balbiani body during early oogenesis of Thermobia.

Oocytes of many invertebrate and vertebrate species contain a characteristic organelle complex known as the Balbiani body (Bb). Until now, three principal functions have been ascribed to this complex: delivery of germ cell determinants and localized RNAs to the vegetal cortex/posterior pole of the oocyte, transport of the mitochondria towards the germ plasm, and participation in the formation of lipid droplets. Here, we present the results of a computer-aided 3D reconstruction of the Bb in the growing oocytes of an insect, Thermobia domestica. Our analyses have shown that, in Thermobia, the central part of each fully developed Bb comprises a single intricate mitochondrial network. This "core" network is surrounded by several isolated bean-shaped mitochondrial units that display lowered membrane potential and clear signs of degeneration. In light of the above results and recent theoretical models of mitochondrial quality control, the role of the Bb is discussed. We suggest that, in addition to the aforementioned functions, the Bb is implicated in the selective elimination of dysfunctional mitochondria during oogenesis.

Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid.

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1, and 4-1BB, in AA-treated cells. We demonstrated that down-regulation of the transcription factor glioma-associated protein 1 (GLI1) in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1, and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and down-regulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability, and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating nuclear translocation of NFATc1, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants impaired the AA-induced apoptosis and down-regulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for future PUFA-based therapeutic approaches.

Stable Chromosome Condensation Revealed by Chromosome Conformation Capture.

Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.

Building an integrated model of chromosome congression.

A universal feature of mitosis is that all chromosomes become aligned at the spindle equator--the halfway point between the two spindle poles--prior to anaphase onset. This migratory event is called congression, and is powered by centromere-bound protein machines called kinetochores. This Commentary aims to document recent advances concerning the two kinetochore-based force-generating mechanisms that drive mitotic chromosome congression in vertebrate cells: depolymerisation-coupled pulling (DCP) and lateral sliding. We aim to explore how kinetochores can 'read-out' their spatial position within the spindle, and adjust these force-generating mechanisms to ensure chromosomes reach, and then remain, at the equator. Finally, we will describe the 'life history' of a chromosome, and provide a working model for how individual mechanisms are integrated to ensure efficient and successful congression.

The Balbiani Ring Story: Synthesis, Assembly, Processing, and Transport of Specific Messenger RNA-Protein Complexes.

Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.

Nuclear organization.

This article discusses three reviews on the theme of nuclear organization.

Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking.

Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1-3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.

The Chironomus tentans genome sequence and the organization of the Balbiani ring genes.

BACKGROUND: The polytene nuclei of the dipteran Chironomus tentans (Ch. tentans) with their Balbiani ring (BR) genes constitute an exceptional model system for studies of the expression of endogenous eukaryotic genes. Here, we report the first draft genome of Ch. tentans and characterize its gene expression machineries and genomic architecture of the BR genes. RESULTS: The genome of Ch. tentans is approximately 200 Mb in size, and has a low GC content (31%) and a low repeat fraction (15%) compared to other Dipteran species. Phylogenetic inference revealed that Ch. tentans is a sister clade to mosquitoes, with a split 150-250 million years ago. To characterize the Ch. tentans gene expression machineries, we identified potential orthologus sequences to more than 600 Drosophila melanogaster (D. melanogaster) proteins involved in the expression of protein-coding genes. We report novel data on the organization of the BR gene loci, including a novel putative BR gene, and we present a model for the organization of chromatin bundles in the BR2 puff based on genic and intergenic in situ hybridizations. CONCLUSIONS: We show that the molecular machineries operating in gene expression are largely conserved between Ch. tentans and D. melanogaster, and we provide enhanced insight into the organization and expression of the BR genes. Our data strengthen the generality of the BR genes as a unique model system and provide essential background for in-depth studies of the biogenesis of messenger ribonucleoprotein complexes.

The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila.

The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate the molecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification.

Light-inducible molecular beacons for spatio-temporally highly defined activation.

We have developed a new molecular beacon design that requires an additional UV pulse for fluorescence activation. This improves the signal-to-noise ratio tremendously compared to previous approaches and allows for a precise control of the time point and location of RNA labelling.

The infant and pubertal human ovary: Balbiani's body-associated VASA expression, immunohistochemical detection of apoptosis-related BCL2 and BAX proteins, and DNA fragmentation.

STUDY QUESTION: How do apoptosis-related BCL2 and BAX genes, known to regulate death or survival of germ cells in fetal and adult life, and germ-cell-specific VASA protein behave from birth to puberty in the human ovary? SUMMARY ANSWER: In resting primordial follicles in both infant and pubertal ovaries, BCL2 family members and germ-cell-specific VASA behave as in fetal life. After birth, once follicles leave the resting reserve to enter the growing follicular pool, detection of apoptosis-related genes moves from the germ cell to granulosa cells and VASA expression is lost. WHAT IS KNOWN ALREADY: In the human ovary, around 85% of the 7 × 106 potential oocytes produced at mid-gestation are lost before birth, an extra 10% before puberty, and loss continues throughout reproductive life until germinal exhaustion of the ovary. Oocyte loss is mainly driven through a balanced expression of BCL2 gene family members. Apoptosis-inducing BAX gene shows a sustained expression throughout fetal and adult life, whereas apoptosis-inhibiting BCL2 is detectable during the proliferative stage of primordial germ cells and oogonia in the fetal ovary and proliferation of granulosa cells in growing follicles in the adult ovary. The germ-cell marker VASA is detectable in the fetal ovary from early oogenesis and is conspicuously expressed in primordial follicles, where in late pregnancy it is associated with the Balbiani's vitelline space. VASA expression is not detectable in the adult ovary. STUDY DESIGN, SIZE, DURATION: This is a qualitative analysis involving infant/pubertal paraffin-embedded human ovaries screened for apoptosis-related proteins, DNA fragmentation and germ-cell identity. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovaries from 13 patients ranging in age from 4 to 16 years, undergoing gynaecological surgical procedures due to benign pathology, were studied. Tissues were fixed in 10% formalin, paraffin-embedded and processed for immunohistochemistry to screen the temporal and cellular localization of germ-cell-specific VASA protein and BCL2 and BAX apoptosis-related proteins. In addition, a terminal deoxynucleotidyl transferase-mediated deoxiuridinetriphosphate nick-end labelling (TUNEL) assay was performed to detect DNA fragmentation. General histology and tissue integrity were assessed by haematoxylin-eosin staining. MAIN RESULTS AND THE ROLE OF CHANCE: VASA showed a differential pattern of expression; in the resting primordial follicle reserve in infant and pubertal ovaries, it was associated with the Balbiani's body space in the germ cell. VASA remained detectable in primary follicles leaving the resting reserve, but once follicles entered the growing pool it became undetectable. This pattern of VASA expression is the same as in the fetal ovary. BAX was expressed both in the resting primordial reserve and in the pool of growing follicles, whereas BCL2 was detected only in granulosa cells in antral follicles in the growing pool. Apoptosis-related protein expression moved from the germ cell to the somatic stratum when primordial follicles left the resting reserve to enter the pool of growing follicles, irrespective of female age. Most TUNEL-positive cells were detected in the granulosa cells of antral follicles. No TUNEL-positive cells were found in resting primordial follicles. LIMITATIONS, REASONS FOR CAUTION: The study was limited by the qualitative nature of the immuno-histochemical analysis and the TUNEL assay. The results neither quantify the levels of germ-cell death nor exclude other concurrent cell death mechanisms that could act in the regulation of female germ-cell number. WIDER IMPLICATIONS OF THE FINDING: This study provides missing knowledge about apoptosis and germ-cell-specific VASA expression in the human ovary between birth and puberty and the participation of BCL2 and BAX genes in the balance between death and survival throughout female germ-line development. Intracellular localization of VASA in resting follicles emerges as a possible marker with prognostic value that needs further investigation, especially in infant patients entering ovarian cryo-preservation programmes. This knowledge will be valuable in optimizing the rescue and clinical use of germ cells to restore fertility in women. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. The authors have no conflicts of interest to declare.

Differential expression of ribosomal protein gene, gonadotrophin releasing hormone gene and Balbiani ring protein gene in silver nanoparticles exposed Chironomus riparius.

The eco- and genotoxicity of silver nanoparticles (AgNPs) was investigated in the fourth instar larvae of the aquatic midge, Chironomus riparius. AgNPs did not have acute toxicity in C. riparius, but did exhibited chronic toxicity on development (pupation and emergence failure) and reproduction. Genotoxicity also occurred in AgNPs exposed C. riparius. Differential Display PCR (DD-PCR), based on the Annealing Control Primer (ACP) technique, was conducted to investigate the underlying toxic mechanism, which identified altered gene expression in C. riparius after treatment with AgNPs. The possible toxicity mechanism of AgNPs in C. riparius involves the down regulation of the ribosomal protein gene (CrL15) affecting the ribosomal assembly and consequently, protein synthesis. Up regulation of the gonadotrophin releasing hormone gene (CrGnRH1) might lead to the activation of gonadotrophin releasing hormone mediated signal transduction pathways and reproductive failure. Up regulation of the Balbiani ring protein gene (CrBR2.2) may be an indication of the organism's protection mechanism against the AgNPs. The overall results suggest that the toxicity of AgNPs towards aquatic organisms should be thoroughly investigated to allow for their safe use, as they seem to exhibit important toxicity towards C. riparius.

Nucleocytoplasmic mRNP export is an integral part of mRNP biogenesis.

Nucleocytoplasmic export and biogenesis of mRNPs are closely coupled. At the gene, concomitant with synthesis of the pre-mRNA, the transcription machinery, hnRNP proteins, processing, quality control and export machineries cooperate to release processed and export competent mRNPs. After diffusion through the interchromatin space, the mRNPs are translocated through the nuclear pore complex and released into the cytoplasm. At the nuclear pore complex, defined compositional and conformational changes are triggered, but specific cotranscriptionally added components are retained in the mRNP and subsequently influence the cytoplasmic fate of the mRNP. Processes taking place at the gene locus and at the nuclear pore complex are crucial for integrating export as an essential part of gene expression. Spatial, temporal and structural aspects of these events have been highlighted in analyses of the Balbiani ring genes.

Balbiani ring mRNPs diffuse through and bind to clusters of large intranuclear molecular structures.

A detailed conception of intranuclear messenger ribonucleoprotein particle (mRNP) dynamics is required for the understanding of mRNP processing and gene expression outcome. We used complementary state-of-the-art fluorescence techniques to quantify native mRNP mobility at the single particle level in living salivary gland cell nuclei. Molecular beacons and fluorescent oligonucleotides were used to specifically label BR2.1 mRNPs by an in vivo fluorescence in situ hybridization approach. We characterized two major mobility components of the BR2.1 mRNPs. These components with diffusion coefficients of 0.3 ± 0.02 µm²/s and 0.73 ± 0.03 µm²/s were observed independently of the staining method and measurement technique used. The mobility analysis of inert tracer molecules revealed that the gland cell nuclei contain large molecular nonchromatin structures, which hinder the mobility of large molecules and particles. The mRNPs are not only hindered by these mobility barriers, but in addition also interact presumably with these structures, what further reduces their mobility and effectively leads to the occurrence of the two diffusion coefficients. In addition, we provide evidence that the remarkably high mobility of the large, 50 nm-sized BR2.1 mRNPs was due to the absence of retarding chromatin.

A direct role for cohesin in gene regulation and ecdysone response in Drosophila salivary glands.

BACKGROUND: Developmental abnormalities observed in Cornelia de Lange syndrome have been genetically linked to mutations in the cohesin machinery. These and other recent experimental findings have led to the suggestion that cohesin, in addition to its canonical function of mediating sister chromatid cohesion, might also be involved in regulating gene expression. RESULTS: We report that cleavage of cohesin's kleisin subunit in postmitotic Drosophila salivary glands induces major changes in the transcript levels of many genes. Kinetic analyses of changes in transcript levels upon cohesin cleavage reveal that a subset of genes responds to cohesin cleavage within a few hours. In addition, cohesin binds to most of these loci, suggesting that cohesin is directly regulating their expression. Among these genes are several that are regulated by the steroid hormone ecdysone. Cytological visualization of transcription at selected ecdysone-responsive genes reveals that puffing at Eip74EF ceases within an hour or two of cohesin cleavage, long before any decline in ecdysone receptor could be detected at this locus. CONCLUSION: We conclude that cohesin regulates expression of a distinct set of genes, including those mediating the ecdysone response.