Baskar Raju, Bakrhat Anna, Abdu Uri
Department of Life Sciences, Ben-Gurion University of the Negev, Be'er Sheva 8410501, Israel.
Department of Life Sciences, Ben-Gurion University of the Negev, Be'er Sheva 8410501, Israel
Biol Open. 2019 Jan 17;8(1):bio039552. doi: 10.1242/bio.039552.
The polarized organization of the oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). In this study, we used the gene , the putative homologue of , to develop a unique genetic reporter for asymmetric oocyte organization. We generated a null allele of the gene using the CRISPR-Cas9 system and found that is not required for determining the axes of the embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, we demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal MT-organizing centers. Our results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity.
通过检查mRNA的不对称定位可以观察到卵母细胞的极化组织，这由极化微管（MTs）网络提供支持。在本研究中，我们使用了基因（推测为某基因的同源物）来开发一种用于卵母细胞不对称组织的独特遗传报告基因。我们使用CRISPR-Cas9系统产生了该基因的无效等位基因，发现其对于确定胚胎的轴并非必需。然而，截短形式的GFP-Forked的异位表达产生了一个独特的不对称Forked网络，其首先在卵母细胞后部积累，然后在卵子发生中期局限于卵母细胞皮质的前外侧区域。这种定位模式类似于报道的极化MTs网络的定位模式。实际上，对卵母细胞极化组织的药理学和遗传学操作表明，丝状Forked网络会扩散到卵母细胞的整个皮质表面，这在卵母细胞极化受到干扰时是可以预期的。最后，我们证明Forked与Short-stop和Patronin焦点相关联，这些焦点组装非中心体MT组织中心。因此，我们的结果表明，不对称GFP-Forked网络定位的清晰可视化可作为研究卵母细胞极性的一种新工具。
