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Background: Changes in transcellular bioelectrical patterns are known to play important roles during developmental and regenerative processes. The Drosophila follicular epithelium has proven to be an appropriate model system for studying the mechanisms by which bioelectrical signals emerge and act. Fluorescent indicator dyes in combination with various inhibitors of ion-transport mechanisms have been used to investigate the generation of membrane potentials (V mem ) and intracellular pH (pH i ). Both parameters as well as their anteroposterior and dorsoventral gradients were affected by the inhibitors which, in addition, led to alterations of microfilament and microtubule patterns equivalent to those observed during follicle-cell differentiation., Results: We expressed two genetically-encoded fluorescent sensors for V mem and pH i , ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on V mem and/or pH i as previously described for V mem - and pH i -sensitive fluorescent dyes. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified exerting influence on ovary development and/or oogenesis. Loss of ovaries or small ovaries were the results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk). Germline knockdown of rpk also resulted in smaller ovaries. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10A onward. In addition, soma knockdown of the open rectifier K + channel 1 (ork1) resulted in a characteristic round-egg phenotype with altered microfilament and microtubule organisation in the follicular epithelium., Conclusions: The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed V mem - and pH i -sensors exhibit some practical advantages compared to fluorescent indicator dyes. Their use confirms that the ion-transport mechanisms targeted by inhibitors play important roles in the generation of bioelectrical signals. Moreover, modulation of bioelectrical signals via RNAi-knockdown of genes coding for ion-transport mechanisms and gap-junction subunits exerts influence on crucial processes during ovary development and results in cytoskeletal changes and altered follicle shape. Thus, further evidence amounts for bioelectrical regulation of developmental processes via the control of both signalling pathways and cytoskeletal organisation.

Background: During Drosophila oogenesis, the follicular epithelium differentiates into several morphologically distinct follicle-cell populations. Characteristic bioelectrical properties make this tissue a suitable model system for studying connections between electrochemical signals and the organisation of the cytoskeleton. Recently, we have described stage-specific transcellular antero-posterior and dorso-ventral gradients of intracellular pH (pH i ) and membrane potential (V mem ) depending on the asymmetrical distribution and/or activity of various ion-transport mechanisms. In the present study, we analysed the patterns of basal microfilaments (bMF) and microtubules (MT) in relation to electrochemical signals., Results: The bMF- and MT-patterns in developmental stages 8 to 12 were visualised using labelled phalloidin and an antibody against acetylated α-tubulin as well as follicle-cell specific expression of GFP-actin and GFP-α-tubulin. Obviously, stage-specific changes of the pH i - and V mem -gradients correlate with modifications of the bMF- and MT-organisation. In order to test whether cytoskeletal modifications depend directly on bioelectrical changes, we used inhibitors of ion-transport mechanisms that have previously been shown to modify pH i and V mem as well as the respective gradients. We inhibited, in stage 10b, Na + /H + -exchangers and Na + -channels with amiloride, V-ATPases with bafilomycin, ATP-sensitive K + -channels with glibenclamide, voltage-dependent L-type Ca 2+ -channels with verapamil, Cl - -channels with 9-anthroic acid and Na + /K + /2Cl - -cotransporters with furosemide, respectively. The correlations between pH i , V mem , bMF and MT observed in different follicle-cell types are in line with the correlations resulting from the inhibition experiments. While relative alkalisation and/or hyperpolarisation stabilised the parallel transversal alignment of bMF, acidification led to increasing disorder and to condensations of bMF. On the other hand, relative acidification as well as hyperpolarisation stabilised the longitudinal orientation of MT, whereas alkalisation led to loss of this arrangement and to partial disintegration of MT., Conclusions: We conclude that the pH i - and V mem -changes induced by inhibitors of ion-transport mechanisms simulate bioelectrical changes occurring naturally and leading to the cytoskeletal changes observed during differentiation of the follicle-cell epithelium. Therefore, gradual modifications of electrochemical signals can serve as physiological means to regulate cell and tissue architecture by modifying cytoskeletal patterns.

In Drosophila ovarian follicles, communication via gap junctions can be observed between the oocyte and its surrounding follicular epithelium. In the present study, the intercellular exchange of the fluorescent tracer Lucifer Yellow was analysed following pressure-injections of five different sera or protein solutions into the oocyte of stage-10 follicles. Three of the tested sera are directed against a channel-forming 16 kDa protein, which is a component of the vacuolar H(+)-ATPase and of Nephrops norvegicus gap junctions. When one of these antisera was injected 5-10 min prior to the dye, the percentage of follicles showing dye-coupling between oocyte and follicle cells was extremely small. On the other hand, injections of non-immune serum or of bovine serum albumin solution had only minor inhibitory effects. With indirect immunofluorescence, the three Nephrops antisera revealed a discrete punctate pattern at the membranes between neighbouring follicle cells as well as between follicle cells and oocyte. Most likely, this fluorescent pattern represents the distribution of gap junctions in the follicular epithelium. On immunoblots, the Nephrops antisera recognized a 29 kDa Drosophila ovary protein with high specificity. Affinity purification of one of these antisera against the 29 kDa protein revealed that this protein of Drosophila and the 16 kDa membrane-channel protein of Nephrops are immunologically related. Thus, the Nephrops antisera might help to reveal, in future injection experiments, the functional role of gap-junction mediated communication in Drosophila.

Charged cell constituents in polytrophic insect follicles are thought to be transported in the nurse cell-oocyte syncytium by way of electrophoresis. This concept, proposed by Woodruff & Telfer (1980) was based on electrophysiological data and microinjection of heterologous proteins using Hyalophora follicles. By microinjecting fluorescently labelled acidic and basic proteins into the nurse cells or oocyte of vitellogenic Drosophila follicles, we failed to obtain evidence for charge-dependent migration of these molecules. We have also analyzed the proteins of nurse cells and oocyte on isoelectric focusing gels, by means of two-dimensional gel electrophoresis, and by ion exchange chromatography to see if basic or acidic proteins accumulate in vivo in nurse cells and oocyte, respectively. For the bulk of the follicular proteins we found no accumulation. Further evidence against an electrophoretic transport system in Drosophila was obtained by estimating the intracellular pH from the colour of indicator dyes microinjected into the follicles; the results indicate that the pH in the nurse cell cytoplasm is lower than that in the ooplasm. According to the model developed for Hyalophora, electrophoretic transport would be favoured by high pH in the nurse cell cytoplasm.