Untersuchungen zur elektrochemischen Regulation der Musterbildung im Ovar von Drosophila melanogaster

  • Investigations on the electrochemical regulation of pattern formation in the ovary of Drosophila melanogaster

Schotthöfer, Susanne Katharina; Bohrmann, Johannes (Thesis advisor); Spehr, Marc (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Several developmental and regenerative processes are known to be controlled by bioelectrical signals. Transcellular changes in membrane potential (Vmem)- and intracellular pH (pHi)-patterns, like electrochemical gradients, affect cytoskeletal organisation and planar cell polarity. The main focus of the present study was the identification of ion-transport mechanisms and signalling pathways that form the basis of this electrochemical regulation, and the evaluation of earlier results using genetic methods. Axis formation is one of several developmental processes for which electrochemical regulation has been demonstrated, and connections between bioelectrical polarity and cytoskeletal polarity are therefore assumed. The present study clearly indicates that bioelectrical polarity and cytoskeletal polarity are closely linked to axial polarity in Drosophila wild-type and gurken (grk) mutant follicles during the course of oogenesis. Corresponding to a disturbed morphological anterior-posterior (a-p) polarity in grk, the follicular epithelium (FE) showed, in stage 9, a significantly reduced a-p Vmem-gradient compared to wild type and changes in cytoskeletal organisation. The most striking differences were visible during stage 10B when dorsal-ventral (d-v) polarity is established. Concurrent with morphological d-v polarity, significant d-v electrochemical gradients and characteristic stage-specific basal microfilament and microtubule patterns emerged in the wild type. In grk, however, comparable transversal electrochemical gradients, characteristic cytoskeletal patterns and a morphological d-v polarity were absent. Presumably, missing Grk-EGFR signalling acts influence on the asymmetric distribution or activation of ion-transport mechanisms and gap junctions. Consequently, electrochemical gradients are influenced, alterations in cytoskeletal organisation fail to occur, and the morphology of the FE changes. Previously identified ion-transport mechanisms in the FE were reevaluated using the genetically-encoded Vmem-sensor ArcLight and the pHi-sensor pHluorin-Moesin in combination with specific inhibitors. The inhibition experiments using the genetically-encoded sensors confirm that the targeted ion-transport mechanisms play important roles in generating bioelectrical signals in the FE. We detected significant Vmem- and pHi-changes which are comparable to previously described changes using the voltage- and pH-sensitive fluorescent dyes DiBAC and CFDA. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified excerting influence on ovary development and/or oogenesis. Complete loss of ovaries or small ovaries were observed as results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk), as well as of germline knockdown of rpk. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10B onward. The highly penetrant knockdown phenotypes suggest that the induced electrochemical dysregulation has massive impact on cytoskeletal organisation. Accordingly, differentiation of somatic stem cells as well as ovary morphology or the development of follicles are disturbed.Comparable to changes in cytoskeletal properties in grk, soma knockdown of the open rectifier K+ channel 1 (ork1) resulted in altered basal microfilament and microtubule patterns. ork1-follicles show a characteristic round-egg phenotype, resembling the phenotype of known round-egg-mutants. Round-egg mutants have been associated with the Fat2 planar cell-polarity pathway in the FE. In summary, the results of this study provide further evidence for electrochemical regulation of developmental processes via the control of signalling pathways and cytoskeletal elements. As the analysis of RNAi-knockdowns and the mutant grk indicate, electrochemical dysregulation affects essential functions during Drosophila ovary development and oogenesis. Changes in bioelectrical properties cause alterations in the organisation of the cytoskeleton, which finally result in altered morphology of the FE and the whole follicle.