Bioelectrical and cytoskeletal patterns correlate with altered axial polarity in the follicular epithelium of the Drosophila mutant gurken

Abstract

Background: Bioelectrical signals are known to be involved in the generation of cell and tissue polarity as well as in cytoskeletal dynamics. The epithelium of Drosophila ovarian follicles is a suitable model system for studying connections between electrochemical gradients, patterns of cytoskeletal elements and axial polarity. By interactions between soma and germline cells, the transforming growth factor-α homolog Gurken (Grk) establishes both the anteroposterior and the dorsoventral axis during oogenesis. Results: In the follicular epithelium of the wild-type (wt) and the polarity mutant grk, we analysed stage-specific gradients of membrane potentials (V mem) and intracellular pH (pH i) using the potentiometric dye DiBAC 4 (3) and the fluorescent pH-indicator 5-CFDA,AM, respectively. In addition, we compared the cytoskeletal organisation in the follicular epithelium of wt and grk using fluorescent phalloidin and an antibody against acetylated α-tubulin. Corresponding to impaired polarity in grk, the slope of the anteroposterior V mem-gradient in stage S9 is significantly reduced compared to wt. Even more striking differences in V mem-and pH i-patterns become obvious during stage S10B, when the respective dorsoventral gradients are established in wt but not in grk. Concurrent with bioelectrical differences, wt and grk exhibit differences concerning cytoskeletal patterns in the follicular epithelium. During all vitellogenic stages, basal microfilaments in grk are characterised by transversal alignment, while wt-typical condensations in centripetal follicle cells (S9) and in dorsal centripetal follicle cells (S10B) are absent. Moreover, in grk, longitudinal alignment of microtubules occurs throughout vitellogenesis in all follicle cells, whereas in wt, microtubules in mainbody and posterior follicle cells exhibit a more cell-autonomous organisation. Therefore, in contrast to wt, the follicular epithelium in grk is characterised by missing or shallower electrochemical gradients and by more coordinated transcellular cytoskeletal patterns. Conclusions: Our results show that bioelectrical polarity and cytoskeletal polarity are closely linked to axial polarity in both wt and grk. When primary polarity signals are altered, both bioelectrical and cytoskeletal patterns in the follicular epithelium change. We propose that not only cell-specific levels of V mem and pH i , or the polarities of transcellular electrochemical gradients, but also the slopes of these gradients are crucial for cytoskeletal modifications and, thus, for proper development of epithelial polarity.

Analysing bioelectrical phenomena in the Drosophila ovary with genetic tools: tissue-specific expression of sensors for membrane potential and intracellular pH, and RNAi-knockdown of mechanisms involved in ion exchange

“…This interpretation is in accordance with previous studies suggesting influences of V mem -and pH i -changes on cytoskeletal organisation and planar cell polarity [5,16]. Moreover, correlations between alterations in bioelectrical patterns and changes in planar cell polarity were recently described in the mutant gurken [17].…”

“…Tangential optical sections of typical S9, S10A, S10B and S12 wt-and ork1-follicles stained with fluorescent phalloidin are shown (cFC, centripetal FC; mFC, mainbody FC; vFC, ventral FC; a, anterior; p, posterior; d, dorsal; v, ventral); d-v orientation, as indicated, applies to all images, except vFC-images (scale bars represent 10 μm). Since, due to bMF-condensations in wt-follicles, it is difficult to reveal transversal bMF-alignment in dorsal and lateral cFC [5,17], vFC are shown for S10B. Typical bMF-condensations (asterisks), as in S9 cFC in wt, are missing in ork1.…”

“…Female flies were killed, 2-3 days old ovaries were dissected, and single follicles of vitellogenic stages S8-S12 were isolated as described previously [5,16,17]. Dissection and cytoskeletal staining were carried out in Drosophila phosphate buffered saline [75], whereas inhibition experiments and morphological analysis were carried out in R-14 Medium [75,76].…”

“…Single follicles of S8-S12, expressing either ArcLight or pHluorin-Moesin, were imaged in R-14 medium on a Zeiss AxioImager.M2 structured illumination microscope (SIM), equipped with a Zeiss ApoTome and a Zeiss AxioCamMRm camera, using a × 20/0.5 objective. Median optical sections were produced, and ImageJ (NIH, USA) was used to generate pseudocolour images as described previously [5,16,17].…”

“…Moreover, in humans, morphological defects caused by mutations in ion-transport mechanisms are associated with so-called channelopathies [8][9][10][11][12]. Accordingly, pre-patterns of membrane potential (V mem ) and intracellular pH (pH i ) are supposed to represent a basis for tissue and organ patterning via the control of planar cell polarity and cytoskeletal organisation [3,5,[13][14][15][16][17].…”