RWTH Aachen University, Institut für Biologie II, Abt. Zoologie und Humanbiologie, Worringerweg 3, 52056, Aachen, Germany.
BMC Dev Biol. 2020 Jul 8;20(1):15. doi: 10.1186/s12861-020-00220-6.
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) and intracellular pH (pH). 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 V and pH, ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on V and/or pH as previously described for V- and pH-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 Kchannel 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 V- and pH-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.
已知细胞间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转运机制抑制剂的组合已被用于研究膜电位（V）和细胞内 pH（pH）的产生。这两个参数及其前后和背腹梯度都受到抑制剂的影响，此外，这还导致微丝和微管模式的改变，与在滤泡细胞分化过程中观察到的改变相当。
我们在果蝇的滤泡上皮中表达了两种用于 V 和 pH 的遗传编码荧光传感器，ArcLight 和 pHluorin-Moesin。通过各自的抑制剂，我们获得了与以前描述的 V-和 pH 敏感荧光染料相当的 V 和/或 pH 影响。在 RNAi 敲低筛选中，鉴定了五个离子转运机制和间隙连接亚基的基因，它们对卵巢发育和/或卵发生有影响。体敲低间隙连接蛋白 inx1 和 inx3 以及 DEG/ENaC 家族成员 ripped pocket（rpk）会导致卵巢缺失或卵巢变小。生殖系敲低 rpk 也会导致卵巢变小。V-ATPase 亚基 vha55 的体敲低会导致从 10A 期开始卵泡退化的卵巢变小。此外，开放整流钾通道 1（ork1）的体敲低会导致滤泡上皮中微丝和微管组织改变的特征性圆形卵表型。
果蝇的遗传工具包为生物电现象的精细和扩展分析提供了手段。与荧光指示剂染料相比，组织特异性表达的 V 和 pH 传感器具有一些实际优势。它们的使用证实，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。此外，通过 RNAi 敲低编码离子转运机制和间隙连接亚基的基因来调节生物电信号会对卵巢发育过程中的关键过程产生影响，并导致细胞骨架变化和卵泡形状改变。因此，更多的证据表明，通过控制信号通路和细胞骨架组织，生物电调节对发育过程具有调节作用。
