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

Abstract

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 RNAiknockdown 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 sizereduced 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.

Molecular and morphological approach to study the innexin gap junctions in Rhynchosciara americana

“…Dm-Inx2 is expressed in somatic and germ cells, as well as Ra-Inx2. The RNAi knockdown of Inx1 and Inx3 in Drosophila ovarian follicles resulted in follicles without ovaries, small ovaries and few follicles [ 74 ]. Recent studies in D. melanogaster ovaries demonstrate that Inx2 and Inx3 act on border cells regulating microtubules, with Inx4 having a similar role in the oocyte.…”

Cytoophidia Maintain the Integrity of Drosophila Follicle Epithelium

“…Both w 1118 and C-terminal mChe-4V5 tagged CTPS knock-in flies out of w 1118 produced in our laboratory were used as wild-type controls unless stated otherwise. The stocks used were: (1) CTPS H335A mutated with mChe-4V5 tagged CTPS Knock-in fly , (2) Actin-Gal4/Cyo (A gift from Guanjun Gao’s lab, ubiquitous expression under strong promoter, a chromosome II insertion balanced over Curly of Oster [ 32 ]), (3) Tj-Gal4 (A gift from Kun Dou’s lab [ 33 , 34 ]), (4) Sp/Cyo; Sb/Tm6B (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Drosophila Resources and Technology Platform), (5) UAS CTPS-mCherry-OE, and UAS CTPS H355A -mCherry-OE [ 27 ].…”

Information integration during bioelectric regulation of morphogenesis in the embryonic frog brain

“…For example, endogenous bioelectric prepatterns have been functionally implicated in the location and size of organs such as the eye 20 , invertebrate wing [21][22][23][24] , vertebrate appendage [25][26][27] , and face [27][28][29] . Bioelectric prepatterns also polarize the left-right [30][31][32][33] and dorso-ventral 34,35 axes, and are involved in size control [36][37][38][39][40] , embryonic compartmentalization [41][42][43][44][45] , and stem cell function [46][47][48] . Transduction mechanisms for bioelectric signals 49 , downstream transcriptional machinery 50 , and interfaces between electrical and mechanical events 51,52 have begun to be characterized.…”