Lighting up mRNA localization in Drosophila oogenesis

Abstract

The asymmetric localization of four maternal mRNAs - gurken, bicoid, oskar and nanos - in the Drosophila oocyte is essential for the development of the embryonic body axes. Fluorescent imaging methods are now being used to visualize these mRNAs in living tissue, allowing dynamic analysis of their behaviors throughout the process of localization. This review summarizes recent findings from such studies that provide new insight into the elaborate cellular mechanisms that are used to transport mRNAs to different regions of the oocyte and to maintain their localized distributions during oogenesis.

Fig. 1.

Localized distributions of grk, bcd, osk and nos mRNAs. (A) Schematic showing grk (pink), bcd (green) and osk (purple) mRNA localization in mid-oogenesis (stage 9). nos mRNA is not yet localized at this stage. The anteroposterior (AP) and dorsoventral (DV) axes are indicated. (B) GFP-Stau (green), as proxy for osk mRNA, at the posterior pole of the oocyte (oo) during mid-oogenesis. GFP-Stau is also detected in the nurse cell (nc) cytoplasm. The actin cytoskeleton is highlighted in red with phalloidin. fc, follicle cells. Orientation is the same as in A. (C-F) Visualization of endogenous mRNAs using the MS2 system: (C) grk and (D) bcd during mid-oogenesis; (E) bcd and (F) nos in late oocytes. Owing to the promoter used, the MCP-GFP and MCP-RFP fusion proteins are expressed in both the nurse cells and follicle cells, whereas the MS2-tagged mRNAs are produced only in the nurse cells. MCP-GFP/RFP that is not bound to mRNA enters both the nurse cell and follicle cell nuclei. Scale bars: 20 μm. Image in B was modified, with permission, from Huynh et al. (Huynh et al., 2004); image in C was modified, with permission, from Jaramillo et al. (Jaramillo et al., 2008); images in D and E are reproduced, with permission, from Weil et al. (Weil et al., 2006). Image in F is courtesy of K. Sinsimer (Princeton University, Princeton, NJ, USA). bcd, bicoid; grk, gurken; GFP, green fluorescent protein; MCP, MS2 coat protein; nos, nanos; osk, oskar; RFP, red fluorescent protein.

Fig. 2.

Fluorescent labeling methods. (A) A construct designed for in vitro transcription of the gene of interest from a bacteriophage promoter (T7 in this example), with the coding region shown in gray and the 3′UTR containing the RNA localization signal in red. Transcription of this construct by T7 polymerase in the presence of a fluorophore-coupled nucleotide produces fluorescently labeled RNA for injection into cultured egg chambers. RNA can be injected directly into the oocyte as illustrated or into the nurse cells. (B) In vivo labeling of endogenous mRNA by the MS2 system. This strategy requires two components: a transgene (transgene 1) that encodes the target RNA with an insertion of tandem copies of the stem-loop binding site for the bacteriophage MS2 coat protein (MCP), shown here in the 3′ UTR, usually under the control of its own promoter (P_x); and a transgene (transgene 2) that encodes a fluorescent protein fused to MCP (GFP is shown here) under the control of a maternally active promoter (P_mat). Transgenic fly lines for each component are crossed together to generate females that express both the tagged RNA and the MCP-GFP protein in their ovarian nurse cells. When the two transgenes are thus coexpressed, the binding of MCP to its recognition motif labels the RNA with GFP. The nuclear localization signal (NLS) in the MCP-GFP fusion protein retains excess unbound protein in the nucleus, reducing cytoplasmic background. Fluorescently labeled mRNA enters the oocyte from the nurse cells (not shown). (C) Transgenic expression of GFP-tagged localization factors. A transgene encoding a localization factor fused to a fluorescent protein (e.g. GFP) under the control of its own or a maternally active promoter. Expression of the transgene in the nurse cells of transgenic females will result in production of the fusion protein in the nurse cells. Colocalization of the fusion protein with the target RNA could occur in the nurse cells or oocyte (not shown), through direct RNA-protein interaction (as shown) or through their co-assembly into a larger RNP.

Fig. 3.

Models for mRNA localization. In all panels, microtubules are shown in brown, nurse cell and follicle cell nuclei in blue. (A) Movements of grk and bcd mRNAs within the nurse cells during mid-oogenesis. Straight arrows indicate directed movement on microtubules, squiggly arrows indicate movement of grk with cytoplasmic flows. (B) Microtubule-dependent transport of grk, bcd and osk mRNAs within the oocyte during mid-oogenesis. The oocyte nucleus is shown in gray. Colored arrows show the directions of RNA movements. (C) Localization of bcd and nos at late stages of oogenesis. Contraction of the nurse cells for dumping is indicated by gray arrows pointing inward; entry of bcd and nos into the oocyte is indicated by large straight arrows. Small green arrows depict transport of bcd on anterior microtubules, curved dark green arrows depict diffusion of nos facilitated by ooplasmic streaming.

Fig. 4.

Maintenance of localized mRNAs. (A) Maintenance of bcd mRNA at the anterior cortex of late oocytes by continual active transport on microtubules (MT), transition to a static actin-dependent anchoring mechanism at the end of oogenesis, and release from the tight cortical anchor at fertilization. (B) Stable, actin-dependent anchoring of nos mRNA and germ plasm (including osk) in late oocytes. (C) Dynein-dependent anchoring of grk mRNA in sponge bodies during mid-oogenesis. The oocyte nucleus is depicted as a blue circle.