piRNA-dependent transcriptional gene silencing during Drosophila oogenesis and embryogenesis

The PIWI-interacting RNA (piRNA) pathway is a small RNA based intracellular immune system protecting animal gonads from the deleterious effects of transposons, thus maintaining transgenerational genome integrity. In Drosophila melanogaster ovaries, piRNA-Piwi complexes localise to the nucleus and scan nascent transcripts for transposon expression by using complementary antisense piRNAs as guides. Following target engagement, the gonad-specific protein Panoramix (Panx) is recruited and induces transcriptional gene silencing (TGS) by connecting to the general chromatin silencing machinery of the cell resulting in changes of the epigenetic chromatin state, thus shutting down transcription. However, whether Panx acts on its own or if other proteins are involved in piRNA-dependent TGS remains unknown. During my PhD I studied the protein-protein interactions of Panx and codiscovered the Panx induced co-transcriptional silencing (PICTS) complex comprised of Panx, Nxf2 and Nxt1. The PICTS complex induces TGS at active transposon insertions in Drosophila ovaries. Furthermore, I studied the effects of epigenetic inheritance of piRNA-Piwi complexes and the PICTS complex during early Drosophila embryogenesis. Piwi showed no zygotic transcription in somatic cells but strong maternal deposition and localised not only to pole cells, the germ line precursors, but was also strongly enriched in somatic nuclei. Additionally, the PICTS complex was both maternally deposited and zygotically transcribed and co-localised with Piwi in somatic nuclei. Several transposons showed strong zygotic expression during early embryogenesis. However, transcriptional gene silencing occurred at individual transposon insertions and repressive chromatin marks accumulated around the genomic location of transposons targeted by maternally deposited piRNAs. Depletion of maternally deposited Piwi resulted in deregulation of transposons and loss of repressive chromatin marks at associated genomic regions. My PhD project uncovered an epigenetic transposon regulatory complex that showed expression not only in gonadal tissue but also in somatic cells during early embryogenesis and revealed a novel function of the piRNA pathway in transposon control by inducing epigenetic chromatin changes during early Drosophila development

Generic theory of active polar gels: a paradigm for cytoskeletal dynamics

We develop a general theory for active viscoelastic materials made of polar filaments. This theory is motivated by the dynamics of the cytoskeleton. The continuous consumption of a fuel generates a non equilibrium state characterized by the generation of flows and stresses. Our theory can be applied to experiments in which cytoskeletal patterns are set in motion by active processes such as those which are at work in cells.Comment: 28 pages, 2 figure

Neuroinflammation-Driven Lymphangiogenesis in CNS Diseases

The central nervous system (CNS) undergoes immunosurveillance despite the lack of conventional antigen presenting cells and lymphatic vessels in the CNS parenchyma. Additionally, the CNS is bathed in a cerebrospinal fluid (CSF). CSF is continuously produced, and consequently must continuously clear to maintain fluid homeostasis despite the lack of conventional lymphatics. During neuroinflammation, there is often an accumulation of fluid, antigens, and immune cells to affected areas of the brain parenchyma. Failure to effectively drain these factors may result in edema, prolonged immune response, and adverse clinical outcome as observed in conditions including traumatic brain injury, ischemic and hypoxic brain injury, CNS infection, multiple sclerosis (MS), and brain cancer. Consequently, there has been renewed interest surrounding the expansion of lymphatic vessels adjacent to the CNS which are now thought to be central in regulating the drainage of fluid, cells, and waste out of the CNS. These lymphatic vessels, found at the cribriform plate, dorsal dural meninges, base of the brain, and around the spinal cord have each been implicated to have important roles in various CNS diseases. In this review, we discuss the contribution of meningeal lymphatics to these processes during both steady-state conditions and neuroinflammation, as well as discuss some of the many still unknown aspects regarding the role of meningeal lymphatics in neuroinflammation. Specifically, we focus on the observed phenomenon of lymphangiogenesis by a subset of meningeal lymphatics near the cribriform plate during neuroinflammation, and discuss their potential roles in immunosurveillance, fluid clearance, and access to the CSF and CNS compartments. We propose that manipulating CNS lymphatics may be a new therapeutic way to treat CNS infections, stroke, and autoimmunity

piRNA-guided co-transcriptional silencing coopts nuclear export factors.

The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing

Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis

The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 min and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells

A signaling cascade including ARID1A, GADD45B and DUSP1 induces apoptosis and affects the cell cycle of germ cell cancers after romidepsin treatment

In Western countries, the incidence of testicular germ cell cancers (GCC) is steadily rising over the last decades. Mostly, men between 20 and 40 years of age are affected. In general, patients suffering from GCCs are treated by orchiectomy and radio- or chemotherapy. Due to resistance mechanisms, intolerance to the therapy or denial of chemo- / radiotherapy by the patients, GCCs are still a lethal threat, highlighting the need for alternative treatment strategies. In this study, we revealed that germ cell cancer cell lines are highly sensitive to the histone deacetylase inhibitor romidepsin in vitro and in vivo, highlighting romidepsin as a potential therapeutic option for GCC patients. Romidepsin-mediated inhibition of histone deacetylases led to disturbances of the chromatin landscape. This resulted in locus-specific histone-hyper- or hypoacetylation. We found that hypoacetylation at the ARID1A promotor caused repression of the SWI/SNF-complex member ARID1A. In consequence, this resulted in upregulation of the stress-sensors and apoptosis-regulators GADD45B, DUSP1 and CDKN1A. RNAi-driven knock down of ARID1A mimicked in parts the effects of romidepsin, while CRISPR/Cas9-mediated deletion of GADD45B attenuated the romidepsin-provoked induction of apoptosis and cell cycle alterations. We propose a signaling cascade involving ARID1A, GADD45B and DUSP1 as mediators of the romidepsin effects in GCC cells

Multiple Particle Tracking and Two-Point Microrheology in Cells

Mechanical stress and stiffness are increasingly recognized to play important roles in numerous cell biological processes, notably cell differentiation and tissue morphogenesis. Little definite is known, however, about how stress propagates through different cell structures or how it is converted to biochemical signals via mechanotransduction, due in large part to the difficulty of interpreting many cell mechanics experiments. A newly developed technique, two-point microrheology (TPM), can provide highly interpretable, quantitative measurements of cells’ frequency-dependent shear moduli and spectra of their fluctuating intracellular stresses. TPM is a non-invasive method based on measuring the Brownian motion of large numbers of intracellular particles using multiple particle tracking. While requiring only hardware available in many cell biology laboratories–a phase microscope and digital video camera, as a statistical technique, it also requires the automated analysis of many thousands of micrographs. Here we describe in detail the algorithms and software tools used for such large-scale multiple particle tracking, as well as common sources of error and the microscopy methods needed to minimize them. Moreover, we describe the physical principles behind TPM and other passive microrheology methods, their limitations, and typical results for cultured epithelial cells

Acute Cerebrovascular Disease in the Young The Stroke in Young Fabry Patients Study

Background and Purpose-Strokes have especially devastating implications if they occur early in life; however, only limited information exists on the characteristics of acute cerebrovascular disease in young adults. Although risk factors and manifestation of atherosclerosis are commonly associated with stroke in the elderly, recent data suggests different causes for stroke in the young. We initiated the prospective, multinational European study Stroke in Young Fabry Patients (sifap) to characterize a cohort of young stroke patients. Methods-Overall, 5023 patients aged 18 to 55 years with the diagnosis of ischemic stroke (3396), hemorrhagic stroke (271), transient ischemic attack (1071) were enrolled in 15 European countries and 47 centers between April 2007 and January 2010 undergoing a detailed, standardized, clinical, laboratory, and radiological protocol. Results-Median age in the overall cohort was 46 years. Definite Fabry disease was diagnosed in 0.5% (95% confidence interval, 0.4%-0.8%; n=27) of all patients; and probable Fabry disease in additional 18 patients. Males dominated the study population (2962/59%) whereas females outnumbered men (65.3%) among the youngest patients (18-24 years). About 80.5% of the patients had a first stroke. Silent infarcts on magnetic resonance imaging were seen in 20% of patients with a first-ever stroke, and in 11.4% of patients with transient ischemic attack and no history of a previous cerebrovascular event. The most common causes of ischemic stroke were large artery atherosclerosis (18.6%) and dissection (9.9%). Conclusions-Definite Fabry disease occurs in 0.5% and probable Fabry disease in further 0.4% of young stroke patients. Silent infarcts, white matter intensities, and classical risk factors were highly prevalent, emphasizing the need for new early preventive strategies

DRhoGEF2 Regulates Cellular Tension and Cell Pulsations in the Amnioserosa during Drosophila Dorsal Closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction