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

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.

Keyphrases: Syncytium, Abscission, Midbody, Abscission, Anillin, Germline development, Incomplete cytokinesis, Midbody, Syncytiogenesis