The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis

Abstract

In Drosophila melanogaster, formation of the axes and the primordial germ cells is regulated by interactions between the germ line-derived oocyte and the surrounding somatic follicle cells. This reciprocal signaling results in the asymmetric localization of mRNAs and proteins critical for these oogenic processes. Mago Nashi protein interprets the posterior follicle cell-to-oocyte signal to establish the major axes and to determine the fate of the primordial germ cells. Using the yeast two-hybrid system we have identified an RNA-binding protein, Tsunagi, that interacts with Mago Nashi protein. The proteins coimmunoprecipitate and colocalize, indicating that they form a complex in vivo. Immunolocalization reveals that Tsunagi protein is localized within the posterior oocyte cytoplasm during stages 1-5 and 8-9, and that this localization is dependent on wild-type mago nashi function. When tsunagi function is removed from the germ line, egg chambers develop in which the oocyte nucleus fails to migrate, oskar mRNA is not localized within the posterior pole, and dorsal-ventral pattern abnormalities are observed. These results show that a Mago Nashi-Tsunagi protein complex is required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells.

Figure 1

The genomic organization and sequence of tsu. (A) Genomic organization of tsu illustrating the relative position of the flanking genes Pmm45A and Mys45A, and the p[tsu⁺] transgene. For each gene the translation initiation (ATG), the translation termination (*), and the direction of transcription (arrows) are indicated. Proximal is to the left and distal to the right. (B) The relative positions of mutations within the tsu gene. The tsu gene with the point of insertion of the EP P element that produced tsu¹, an allele (tsu⁵) induced by excision of the P element identified in tsu¹, and the EMS-induced tsu² allele. (C) Sequence of the Drosophila tsunagi gene. Lowercase bold letters in the 5′ end indicate the predicted promoter. An uppercase bold G designates the transcription start site. At the 3′ end, the asterisk defines the position of the translational stop site, and the bold uppercase letters show the position of the polyadenylation site. Intronic sequence is illustrated as lowercase letters nested between uppercase letters. Mutations tsu¹ and tsu⁵ (filled triangle); tsu² (#). (D) Amino acid sequence alignment of Drosophila Tsunagi with related proteins from yeast to humans. Amino acid residues are indicated as follows: identical amino acid residues are outlined in black, conserved amino acids are shaded, (∼) are amino acid residues that are absent, and dots represent gaps. The proteins are designated as follows: (RBM8) Homo sapiens; (MmTsu) Mus musculus; (DrTsu) Danio rerio; (Y14) Xenopus laevis; (DmTsu) Drosophila melanogaster; (CeTsu) Caenorhabditis elegans; (SpTsu) Schizosaccharomyces pombe.

Figure 1

The genomic organization and sequence of tsu. (A) Genomic organization of tsu illustrating the relative position of the flanking genes Pmm45A and Mys45A, and the p[tsu⁺] transgene. For each gene the translation initiation (ATG), the translation termination (*), and the direction of transcription (arrows) are indicated. Proximal is to the left and distal to the right. (B) The relative positions of mutations within the tsu gene. The tsu gene with the point of insertion of the EP P element that produced tsu¹, an allele (tsu⁵) induced by excision of the P element identified in tsu¹, and the EMS-induced tsu² allele. (C) Sequence of the Drosophila tsunagi gene. Lowercase bold letters in the 5′ end indicate the predicted promoter. An uppercase bold G designates the transcription start site. At the 3′ end, the asterisk defines the position of the translational stop site, and the bold uppercase letters show the position of the polyadenylation site. Intronic sequence is illustrated as lowercase letters nested between uppercase letters. Mutations tsu¹ and tsu⁵ (filled triangle); tsu² (#). (D) Amino acid sequence alignment of Drosophila Tsunagi with related proteins from yeast to humans. Amino acid residues are indicated as follows: identical amino acid residues are outlined in black, conserved amino acids are shaded, (∼) are amino acid residues that are absent, and dots represent gaps. The proteins are designated as follows: (RBM8) Homo sapiens; (MmTsu) Mus musculus; (DrTsu) Danio rerio; (Y14) Xenopus laevis; (DmTsu) Drosophila melanogaster; (CeTsu) Caenorhabditis elegans; (SpTsu) Schizosaccharomyces pombe.

Figure 2

Tsunagi and Mago form an in vivo complex. (A) Tsunagi protein is detected during all stages of Drosophila development. Affinity-purified anti-Tsunagi rabbit polyclonal antisera recognize a 19-kD protein that matches the size predicted from sequencing of the tsu gene. Protein extracts were resolved on a 10%–20% SDS-PAGE gradient gel. (B) Tsunagi coimmunoprecipitates with Myc-tagged Mago (Myc-Mago). Extracts from wild-type (OreR) or transgenic flies expressing Myc-Mago were immunoprecipitated with the anti-Myc Mab 9E10. Immunoprecipitated proteins were resolved on a 10%–20% SDS-PAGE gradient gel, and the presence of Myc-Mago (left blot) and Tsunagi (right blot) was determined by probing immunoblots with the indicated antisera.

Figure 3

Tsunagi and Mago colocalize during oogenesis. In all panels the oocyte nucleus is marked with an asterisk, protein localized in the posterior pole by an arrow, and follicle cell nuclei by double arrowheads. The anterior sides of the egg chambers are to the left and the posterior to the right in A–D. Tsunagi is in red and GFP–Mago in green. (A) The distribution of Tsunagi (red) in a stage-4 egg chamber. (B) Tsunagi in the follicle-cell nuclei of a stage-10 egg chamber. The spot within the follicle-cell nucleus without detectable Tsunagi is the nucleolus. (C–D) The distribution of GFP–Mago in the same egg chambers as in A and B. (E) Confocal image of a stage-9 egg chamber illustrating the distribution of Tsunagi and posterior at the bottom.

Figure 4

Posterior pole localization of Tsunagi requires mago⁺ function. In all panels arrowheads designate the position of the oocyte nucleus and arrows the posterior pole. Egg chambers are oriented with the anterior to the left and posterior to the right. (A) Confocal image of a wild-type stage-2 egg chamber. At stage 2 Tsunagi is detected within the posterior pole but not within the oocyte nucleus. (B) A confocal image of a stage-2 egg chamber from a hemizygous mago¹ female. Tsunagi is detected in the oocyte nucleus and is not observed above background in the cytoplasm. (C) Epifluorescence of GFP–Mago in a wild-type stage-4 egg chamber. (D) An epifluorescence image of GFP–Mago in a stage-4 egg chamber from a tsu⁷ homozygous female. The distribution of Mago is indistinguishable from its distribution in wild-type egg chambers.

Figure 5

The wild-type function of tsu is required within the germ line for DV patterning and osk mRNA localization during oogenesis. In all panels anterior is to the left and posterior to the right. (A,E) Micrographs of wild-type and mutant tsu (from tsu⁶/tsu⁷ females) eggshells, respectively. (B,F) The distribution of Kinesin::β-gal in wild-type and mutant tsu stage-9 egg chambers (from tsu⁶/tsu⁷ females), respectively. (C,G) The distribution of Nod::β-gal in wild-type and mutant tsu stage-9 egg chambers (from tsu⁶/tsu⁷ females), respectively. (D,H) The position of the oocyte nucleus (green) and the distribution of Gurken protein (red) in stage-10 egg chambers from wild-type and tsu⁶/tsu⁷ females. In each panel the position of the oocyte nucleus is indicated by the arrow and was determined using α-cyclin E. (I) Distribution of grk mRNA in wild-type stage-8 (left) and stage-10 (right) egg chambers. (J) In egg chambers from tsu⁶/tsu⁷ females, grk mRNA is clearly detectable at stage 9 (left) but by stage 10 (right) it is significantly reduced relative to wild type in ∼70% (n = 70) of the chambers. (K,L) In stage-9 and stage-10 egg chambers the oocyte nucleus is mislocalized in ∼20% (n = 114) of the egg chambers. The micrographs show the position of the oocyte nucleus in a stage-9 egg chamber from a tsu⁷ homozygous mother and the distribution of grk mRNA. (L) A high magnification image of the posterior of the stage-9 egg chamber illustrated in K to show the oocyte nucleus.

Figure 6

Germ-line and follicle-cell clonal analysis of tsu. Egg chambers are oriented with anterior to the left and posterior to the right. In B–E the arrowheads indicate the accumulation of osk mRNA. (A) Immunolocalization of Tsunagi in a stage-9 egg chamber showing a tsu¹/tsu¹ follicle-cell clone with its boundaries delineated by the arrows. (B,C) The distribution of osk mRNA in wild-type stage-9 and stage-10 egg chambers, respectively. (D,E) The distribution of osk mRNA in an ovariole derived from a germ-line mosaic female. As seen in D, osk mRNA accumulates in the anterior pole of stage-9 mosaic egg chambers. Posterior pole accumulation of osk mRNA is not detected in mosaic stage 10 (E) egg chambers.