Published On: Thu, Jan 17th, 2019

GFP-Forked, a genetic reporter for studying Drosophila oocyte polarity [METHODS AND TECHNIQUES]

INTRODUCTION

Correct localization of intracellular messenger RNAs (mRNAs) encoding morphogenetic proteins to their distinct subcellular domains are crucial for specification of the body axes of the Drosophila embryo. The roles of three major asymmetrically localized mRNAs, gurken (grk), bicoid (bcd) and oskar (osk), in the process of establishing axial patterning of the oocyte and embryo in mid-oogenesis were clearly established. grk is localized around the oocyte nucleus and determines the dorsal–ventral axis of the oocyte and embryo (Gonzalez-Reyes et al., 1995; Neuman-Silberberg and Schupbach, 1993, 1996; Roth et al., 1995), whereas bcd is localized to the extreme anterior of the oocyte and determines the anterior pattern of the embryo upon translation (Driever and Nusslein-Volhard, 1988a,b). At the same time, osk is localized to the posterior of the oocyte and initiates the development of future germ cells and the embryo abdomen (Ephrussi et al., 1991).

Asymmetric mRNA localization during mid-oogenesis depends on microtubules (MTs), actin networks and motor proteins. During mid-oogenesis (i.e. stages 9 and 10), MTs within the oocyte are organized asymmetrically, with non-centrosomal MT-organization centers (ncMTOCs) being localized solely to the anterior and lateral cortexes of the oocyte (Huynh and St Johnston, 2004; Nashchekin et al., 2016; Theurkauf, 1994). Regulation of the asymmetrical oocyte MT network is mainly controlled by two sequential processes. Initially, an MTOC positioned at the posterior end of the oocyte, close to the nucleus, which is in a symmetric position at this stage, is established. Signaling from grk to EGF receptors at the posterior end of the oocyte (stage 6–7) establishes posterior follicle cell fate (Gonzalez-Reyes et al., 1995; Roth et al., 1995). Subsequently, an unknown signal produced by posterior follicle cells leads to the establishment of Par-1 kinase activity in the posterior oocyte cortex, thereby defining the antero-lateral versus posterior cortical domain in the oocyte (Doerflinger et al., 2006; Shulman et al., 2000). In addition, the same unknown signal triggers disassembly of the posterior MTOC. At the same time, nucleation of new MTs at the oocyte anterior end results in a reversal of polarity within the oocyte (Gonzalez-Reyes et al., 1995; Roth et al., 1995; Theurkauf et al., 1992). Par-1 kinase now restricts the polarization of MTs to the antero-lateral region of the oocyte by suppressing MT nucleation at the oocyte posterior end (Doerflinger et al., 2006; Parton et al., 2011).

Visualization of the asymmetric organization of the Drosophila oocyte is achieved either by in situ hybridization or by staining with antibodies directed against several localized mRNA, such as grk, bcd and osk. In addition, the polarized organization of oocyte MTs can be demonstrated either by staining using anti-tubulin antibodies or by the expression of MT-associated proteins fused to GFP (Parton et al., 2011). Detection of the polarized MT network using anti-tubulin antibodies, however, requires the use of a special protocol (Legent et al., 2015).

In studying the role of forked gene, the Drosophila homologue of espin, in oogenesis, we discovered that ectopic expression of GFP-Forked protein could be used as a novel tool for analyzing oocyte polarity. First, we demonstrated that oocytes containing mutations in forked showed no defects in polarity. On the other hand, upon over-expression of GFP-Forked, the protein first accumulated at the oocyte posterior end and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. We showed that this unique asymmetric localization of GFP-Forked depends on the polarized MT network. We further found that ectopic expression of Forked is associated with Short-stop and Patronin foci. Thus, our results reveal that the novel asymmetric Forked network can be used as a genetic reporter for visualizing and studying oocyte polarity.

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