Evidence for a transport-trap mode of Drosophila melanogaster gurken mRNA localization

Abstract

The Drosophila melanogaster gurken gene encodes a TGF alpha-like signaling molecule that is secreted from the oocyte during two distinct stages of oogenesis to define the coordinate axes of the follicle cell epithelium that surrounds the oocyte and its 15 anterior nurse cells. Because the gurken receptor is expressed throughout the epithelium, axial patterning requires region-specific secretion of Gurken protein, which in turn requires subcellular localization of gurken transcripts. The first stage of Gurken signaling induces anteroposterior pattern in the epithelium and requires the transport of gurken transcripts from nurse cells into the oocyte. The second stage of Gurken signaling induces dorsovental polarity in the epithelium and requires localization of gurken transcripts to the oocyte's anterodorsal corner. Previous studies, relying predominantly on real-time imaging of injected transcripts, indicated that anterodorsal localization involves transport of gurken transcripts to the oocyte's anterior cortex followed by transport to the anterodorsal corner, and anchoring. Such studies further indicated that a single RNA sequence element, the GLS, mediates both transport steps by facilitating association of gurken transcripts with a cytoplasmic dynein motor complex. Finally, it was proposed that the GLS somehow steers the motor complex toward that subset of microtubules that are nucleated around the oocyte nucleus, permitting directed transport to the anterodorsal corner. Here, we re-investigate the role of the GLS using a transgenic fly assay system that includes use of the endogenous gurken promoter and biological rescue as well as RNA localization assays. In contrast to previous reports, our studies indicate that the GLS is sufficient for anterior localization only. Our data support a model in which anterodorsal localization is brought about by repeated rounds of anterior transport, accompanied by specific trapping at the anterodorsal cortex. Our data further indicate that trapping at the anterodorsal corner requires at least one as-yet-unidentified gurken RLE.

Figure 1. Conservation and predicted secondary structure of the GLS.

(A) Sequence alignment of the gurken transcription unit displayed using the Vista Browser at http://pipeline.lbl.gov/cgi-bin/gateway2 . The estimated years in millions (MYA) of evolution between D. melanogaster and each of the other five species is from Heger and Ponting . The most highly conserved region is circled and includes the first 39 nt of the GLS. The last 25 nt of the GLS map to the 3′ side of the abutting intron. The arrow indicates the direction of transcription. The red shaded region corresponds to a putative transposable element. The numbers at the bottom of the graph indicate nucleotide position along the chromosome. (B) The 5′ end of the gurken mRNA, where the green dot denotes the translation start site, the red arrows the boundaries of the GLS, and the asterisk the position of the intron. The nucleotides beneath the aligned sequence blocks highlight differences between the D. Willistoni and D. melanogaster sequences. (C) Predicted secondary structure of the GLS, with non-conserved residues shown in red.

Figure 2. The GLS is sufficient for anterior, but not anterodorsal localization within the Drosophila oocyte.

RNA distribution patterns of wild-type gurken transcripts (A) and K10::GFP reporter transcripts (B–G) as revealed by wholemount in situ hybridization (see Methods). Individual ovarioles are shown, with older egg chambers oriented to the right. The transgenes (B–G) are noted in the individual panels. The structure of the transgenes and expression summary is shown beneath the in situs.

Figure 3. Structure of GLS variants.

The wild-type GLS is shown at the left for comparison. The GLS mutant (referred to as grkGLS^mut in Text) contains 12 point mutations (shown in red), which are predicted to disrupt the predicted base pairing pattern of the GLS at five sites (circled). None of the 12 mutations affect the protein coding sequence as shown at the bottom portion of the figure.

Figure 4. The GLS is required for gurken RNA localization and gene function.

(A–B) Wild-type expression patterns of endogenous gurken RNA (A) and protein (B) as revealed by whole mount in situ hybridization and immunofluorescence, respectively. Anterodorsal localization of transcripts and protein is only apparent in the rightmost egg chambers, which are stage 8 and 9, respectively. (C–E) The gurken RNA and protein distribution patterns of gurken null mutants (grk^ΔFRT) carrying the wild-type gurken transgene, grk^wt (C–D) or no transgene (E). (F–H) grk^ΔFRT eggs and egg chambers (from gurken null mothers) carrying the grkGLS^mut transgene. (F) Left panel: representative grk^ΔFRT; grkGLS^mut egg exhibiting a completely ventralized phenotype, i.e., complete loss of dorsal appendage material. Right panel; anterior end of a grk^ΔFRT; grkGLS^mut egg exhibiting a strong, but not complete, ventralized phenotype. Note, for example the short, fused dorsal appendage. (G) grk^ΔFRT; grkGLS^mut ovariole following in situ hybridization with gurken probe. Transcripts are dispersed throughout the germ-line cysts with only slight enrichment in the oocyte and no subcellular localization. (H) grk^ΔFRT; grkGLS^mut ovariole following immunofluorescence using an anti-Grk antibody. The protein is generally dispersed throughout the germ-line cysts, although slight enrichment around the oocyte nucleus is seen in rare stage 10 and 11 egg chambers.