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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.
BMC Developmental Biology ( IF 1.978 ) Pub Date : 2020-07-08 , DOI: 10.1186/s12861-020-00220-6
Susanne Katharina Schotthöfer 1 , Johannes Bohrmann 1
Affiliation  

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 (Vmem) and intracellular pH (pHi). 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. We expressed two genetically-encoded fluorescent sensors for Vmem and pHi, ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on Vmem and/or pHi as previously described for Vmem- and pHi-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. The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed Vmem- and pHi-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.

中文翻译:

使用遗传工具分析果蝇卵巢中的生物电现象:膜电位和细胞内pH值传感器的组织特异性表达,以及参与离子交换的RNAi敲除机制。

跨细胞生物电模式的变化在发育和再生过程中起着重要作用。果蝇滤泡上皮已被证明是研究生物电信号产生和作用机制的合适模型系统。荧光指示剂染料与各种离子迁移机制抑制剂的组合已用于研究膜电位(Vmem)和细胞内pH(pHi)的产生。抑制剂均会影响这两个参数以及其前后,后腹的梯度,进而导致微丝和微管模式的改变与卵泡细胞分化过程中观察到的改变相同。我们针对Vmem和pHi,ArcLight和pHluorin-Moesin表达了两种经过基因编码的荧光传感器,在果蝇的卵泡上皮中。通过相应的抑制剂,我们获得了对Vmem和/或pHi相当的效果,如先前针对Vmem和pHi敏感的荧光染料所述。在RNAi击倒筛选中,确定了五个离子转运机制和间隙连接亚基的基因,这些基因对卵巢的发育和/或卵子形成有影响。卵巢丢失或小卵巢丢失是体内毒素(innexins)inx1和inx3,以及DEG / ENaC家族成员的口袋撕裂(rpk)导致的体细胞敲除的结果。Rpk的种系敲除也导致较小的卵巢。V-ATPase-亚基vha55的体细胞敲除导致卵巢缩小,卵巢从10A期开始退化。此外,开放整流器K +通道1(ork1)的体细胞敲低导致卵泡上皮细胞中微丝和微管组织改变的特征性圆形蛋表型。果蝇的遗传工具箱提供了对生物电现象进行精细和扩展分析的手段。与荧光指示剂染料相比,组织特异性表达的Vmem和pHi传感器表现出一些实际优势。它们的使用证实了抑制剂靶向的离子转运机制在生物电信号的产生中起着重要作用。而且,通过RNAi敲低编码离子转运机制和间隙连接亚基的基因对生物电信号的调节,对卵巢发育过程中的关键过程产生影响,并导致细胞骨架的变化和卵泡形状的改变。从而,
更新日期:2020-07-08
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