Biogenesis of the C. elegans germline syncytium: from nucleation to maturation

La vie commence par la fusion des gamètes pour générer un zygote, dans lequel les constituants à la fois de l'ovocyte et des spermatozoïdes sont partagés au sein d'un syncytium. Le syncytium consiste en des cellules ou tissus dans lesquels des cellules nucléées individuelles distinctes partagent un cytoplasme commun. Alors que l’avantage du syncytium durant la fécondation est tout à fait évident, les syncytia se produisent également dans de nombreux contextes de développement différents dans les plantes, les champignons et dans le règne animal, des insectes aux humains, pour des raisons qui ne sont pas immédiatement évidentes. Par exemple, la lignée germinale de nombreuses espèces de vertébrés et d'invertébrés, des insectes aux humains, présente une structure syncytiale, suggérant que les syncytia constituent des phases conservées de développement de la lignée germinale. Malgré la prévalence commune des syncytia, ces derniers ont cependant confondu les scientifiques depuis des décennies avec des questions telles que la façon dont ils sont formés et maintenus en concurrence avec leurs homologues diploïdes, et quels sont les avantages et les inconvénients qu'ils apportent. Cette thèse va décrire l'utilisation de la lignée germinale syncytiale de C. elegans afin d'approfondir notre compréhension de l'architecture, la fonction et le mode de formation des tissus syncytiaux. Les cellules germinales (CGs) dans la lignée germinale de C. elegans sont interconnectées les unes aux autres par l'intermédiaire de structures appelées des anneaux de CG. En utilisant l'imagerie des cellules vivantes, nous avons d'abord analysé l'architecture syncytiale de la lignée germinale au long du développement et démontré que la maturation de l'anneau de CG se produit progressivement au cours de la croissance des larves et que les anneaux de CG sont composés de myosine II, de l'anilline canonique ANI-1, et de la courte isoforme d’anilline ANI-2, qui n'a pas les domaines de liaison à l’actine et à la myosine, depuis le premier stade larvaire, L1. Parmi les composants de l'anneau de CG, ANI-2 est exprimé au cours du développement et exclusivement enrichi entre les deux CGs primordiales (CGPs) au cours de l'embryogenèse de C. elegans, indiquant qu’ANI-2 est un composant bona fide des anneaux de CG. Nous avons en outre montré que les anneaux de CG sont largement absents dans les animaux mutants pour ani-2, montrant que leur maintien repose sur l'activité d'ANI-2. Contrairement à cela, nous avons trouvé que la déplétion d’ANI-1 a augmenté à la fois le diamètre des anneaux de CG et la largeur du rachis. Fait intéressant, la déplétion d’ANI-1 dans les mutants d’ani-2 a sauvé les défauts d'anneaux de CG des gonades déficientes en ani-2, ce qui suggère que l'architecture syncytiale de la lignée germinale de C. elegans repose sur un équilibre de l'activité de ces deux protéines Anilline. En outre, nous avons montré que lors de leur entrée à l'âge adulte, les mutants ani-2 présentent de sévères défauts de multinucléation des CGs qui découlent de l'effondrement des membranes de séparation des CGs individuelles. Cette multinucléation a coïncidé avec le début de la diffusion cytoplasmique, dont le blocage réduit la multinucléation des gonades mutantes pour ani-2, suggérant que les anneaux de CG résistent au stress mécanique associé au processus de diffusion cytoplasmique. En accord avec cela, nous avons trouvé aussi que la gonade peut soutenir la déformation élastique en réponse au stress mécanique et que cette propriété repose sur la malléabilité des anneaux de CGs. Dans une étude séparée afin de comprendre le mécanisme de formation du syncytium, nous avons suivi la dynamique de division de la cellule précurseur de la lignée germinale, P4 en deux CGP dans l’embryon de C. elegans. Nous avons démontré que les CGPs commencent la cytocinèse de manière similaire aux cellules somatiques, en formant un sillon de clivage, qui migre correctement et transforme ainsi l'anneau contractile en anneau de « midbody ring » (MBR), une structure qui relie de manière transitoire les cellules en division. Malgré cela, les CGPs, contrairement à leurs homologues somatiques, ne parviennent pas à accomplir la dernière étape de la cytocinèse, qui est la libération abscission-dépendante du MBR. Au lieu de cela, le MBR persiste à la frontière entre les CGPs en division et subit une réorganisation et une maturation pour se transformer finalement en structures en forme d'anneau qui relient les cellules en division. Nous montrons en outre que les composants du MB/MBR; UNC-59Septin, CYK-7, ZEN-4Mklp1, RHO-1RhoA sont localisés à des anneaux de CG au long du développement de la lignée germinale du stade L1 à l'âge adulte, ce qui suggère que les anneaux de CG sont dérivés des MBR. Bien qu'il reste encore beaucoup à faire pour comprendre pleinement le mécanisme précis de la formation du syncytium, le maintien, ainsi que la fonction du syncytium, nos résultats appuient un modèle dans lequel la stabilisation du MBR et la cytocinèse incomplète pourraient être une option conservée dans l’évolution pour la formation du syncytium. En outre, notre travail démontre que les régulateurs de la contractilité peuvent jouer un rôle dans la maturation et l’élasticité de l'anneau de CG au cours du développement de la lignée germinale, fournissant un ajout précieux pour une plus ample compréhension de la syncytiogenèse et de sa fonction.Life begins by the union of oocyte and sperm to generate a zygote, in which the constituents of both gametes are shared within a single cytoplasm in a syncytium. Syncytium is referred to cells or tissues wherein discrete single nucleated cells share a common cytoplasm. While the purpose of a syncytium in fertilization is quite evident, syncytia occur in many different developmental settings in plants, fungi and throughout the animal kingdom for reasons that are not immediately obvious. For instance, germline of many vertebrate and invertebrate species, from insects to humans exhibit syncytial structure, suggesting that syncytia are conserved phase of germline development. Despite the common prevalence of syncytia however, syncytia have confounded scientist for decades with questions such as how they are formed and maintained in competition with their diploid counterparts, and what advantages and disadvantages they bear. This thesis will describe the use of the C. elegans syncytial germline to further our understanding of the architecture, function and mode of formation of the syncytial tissues. Germ cells (GCs) in germline of C. elegans are interconnected to one another via structures here referred to as GC rings. Using live-cell imaging, we first analyzed the germline syncytial architecture throughout development and demonstrated that GC ring maturation occurs progressively during larval growth and that the GC rings are composed of Myosin II, the canonical anillin ANI-1 and ANI-2 the short isoform of anillin that lacks the actin- and myosin- binding domains, since the first larval stage, L1. Among GC ring components, ANI-2 is developmentally expressed and exclusively enriched between the two primordial GCs (PGCs) during C. elegans embryogenesis, indicating that ANI-2 is a bona fide component of GC rings. We further showed that the GC rings are largely absent in ani-2 mutant animals, showing that their maintenance relies on the activity of ANI-2. Contrary to this, we found that ANI-1 depletion increased both the diameter of GC rings and the width of the rachis. Interestingly, depletion of ANI-1 partially rescued the GC ring defects of ani-2-deficient gonads, suggesting that the C. elegans germline syncytial architecture relies on a balance between activities of these two Anillin proteins. Moreover, we showed that adult ani-2 mutants exhibit severe GC multinucleation defects that arise from a collapse of the membranes separating individual GCs. This GC multinucleation initiated at the transition from L4 to adult, which coincided with the onset of oogenesis and cytoplasmic streaming in the rachis. We found that multinucleation is dependent on oogenesis, as GC multinucleation was reduced in conditions where oogenesis was absent. In consistent with this, we further found that the gonad can sustain elastic deformation in response to mechanical stress and that this property relies on malleability of GC rings provided by ANI-2. In a separate study to understand the mechanism of syncytium formation, we monitored the dynamics of the germline founder cell (P4) cytokinesis into Z2 and Z3 during embryogenesis. We found that P4 accomplishes the first phase of cytokinesis, cytoplasmic isolation. In support of this, we found that there is no cytoplasmic exchange of a fluorescent marker between Z2 and Z3 shortly after birth, suggesting that they are not syncytial at this stage. Interestingly however, P4 fails to complete the last phase of cytokinesis, abscission wherein the midbody-ring (MBR) is released from the cell-cell boundary and eventually disappears. Instead, the MBR connecting Z2 and Z3 remains tightly associated to the cortex throughout embryogenesis, forming a stable structure. Interestingly, we found that components of persisting MBRs are all stable constituents of GC rings of the syncytial gonad, suggesting that GC rings are derived from stabilized MBRs. While much remains to be done to fully understand the precise mechanism of syncytium formation, maintenance and function, our findings support a model in which MBR stabilization and incomplete cytokinesis could be an evolutionary conserved feature for syncytium formation. In addition, our work demonstrates that contractility regulators may play a role in GC ring maturation and GC ring elasticity during germline development, providing a valuable addition for further understanding syncytiogenesis and its function

A Multidomain Model for Ionic Electrodiffusion and Osmosis with an Application to Cortical Spreading Depression

Ionic electrodiffusion and osmotic water flow are central processes in many physiological systems. We formulate a system of partial differential equations that governs ion movement and water flow in biological tissue. A salient feature of this model is that it satisfies a free energy identity, ensuring the thermodynamic consistency of the model. A numerical scheme is developed for the model in one spatial dimension and is applied to a model of cortical spreading depression, a propagating breakdown of ionic and cell volume homeostasis in the brain.Comment: submitted for publication, Aug. 28, 201

Investigation of Apoptosis and Metabolism as Mechanisms of Trichloroethylene Toxicity During Pregnancy

Trichloroethylene (TCE), an organic solvent with multiple industrial uses, is associated with adverse pregnancy outcomes in epidemiological studies. In timed-pregnant Wistar rats, TCE exposure reduces fetal weight. Additionally, the TCE metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) stimulates reactive oxygen species (ROS) generation and apoptosis in HTR-8/SVneo placental cells as mechanisms of toxicity. By using aminooxyacetic acid (AOAA) to decrease cysteine conjugate beta-lyase (CCBL) activity and resulting toxicity of DCVC, prior studies in kidney cells demonstrated that the metabolism of DCVC into 1,2-dichlorovinylthiol (DCVT) by CCBL is critical for producing cell death. N-acetyl-L-cysteine (NAC) is a compound with ROS-scavenging properties and a supplier of an acetyl group that contributes to N-acetylation of DCVC. Because metabolism of DCVC is crucial for TCE toxicity, this dissertation hypothesizes that modification of TCE or DCVC metabolism during pregnancy modulates TCE or DCVC-stimulated apoptosis and toxicity. This dissertation uses AOAA and NAC as modulators of TCE or DCVC-stimulated toxicity. Chapter 2 described TCE-induced decreased fetal weight in timed-pregnant Wistar rats that was prevented by AOAA but not NAC pre/co-treatment. However, AOAA reduced CCBL activity in maternal kidney, but not maternal liver or placenta, suggesting that inhibition of kidney metabolism may have a role in the AOAA effect. Morphometric analysis of the placenta indicated that NAC pre/co-treatment with TCE relative to TCE treatment alone altered placental dimensions consistent with a delayed developmental phenotype. Treatments failed to stimulate any indication of placental apoptosis. Chapter 3 findings showed that TCE exposure stimulates changes in amniotic fluid of the rats. Changes in amniotic fluid of both sexes include decreased adenosine triphosphate, decreased adenosine diphosphate, decreased guanosine diphosphate, and altered pentose phosphate pathway and folate biosynthesis. Chapter 4 described mechanisms of DCVC-stimulated toxicity in the in vitro human trophoblast BeWo cell model. BeWo cells are known to syncytialize, or multinucleate and fuse, in response to forskolin to create the syncytiotrophoblast cell type that is the maternal-fetal interface in vivo. DCVC was found to increase caspase 3/7 activity and nuclear condensation or fragmentation, markers of apoptosis, in unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells. DCVC also stimulated changes consistent with increased ROS production in all three BeWo cell types, including increased hydrogen peroxide abundance and decreased PRDX2 mRNA expression. Chapter 5 showed NAC and AOAA pre/co-treatment modulation of DCVC-stimulated toxicity. NAC either exacerbated or did not affect the DCVC-stimulated response. Because NAC exerted a significant effect on increased CYP3A4 mRNA expression, it is possibile that NAC could have contributed to the CYP3A4-generation of the toxic N-acetyl DCVC sulfoxide (NAcDCVCS) metabolite. AOAA did not modify CCBL activity in BeWo cells regardless of differentiation status. However, syncytialized BeWo cells exhibited higher CCBL activity than unsyncytialized BeWo cells, which could explain increased susceptibility of syncytialized BeWo to DCVC apoptosis compared with unsyncytialized BeWo cells. Chapter 6 revealed that DCVC treatment during syncytialization could either exacerbate or reverse some cellular energy metabolism changes stimulated by syncytialization by itself. Notable reversal effects included ratios within purine metabolism. This research indicates a critical role for CCBL in DCVC- and TCE-stimulated toxicity during pregnancy and raises the possibility that NAC pre/co-treatment with TCE or DCVC could be detrimental. Apoptosis and disrupted energy metabolism could be major mechanisms of TCE or DCVC-stimulated toxicity. Important future work includes detection of reactive TCE metabolites and identification of enzyme targets of TCE.PHDToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163113/1/ansu_1.pd