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aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIF2C

¹ Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
² Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA

View larger version (22K): [in a new window]   Fig. 1. Identification of aubergine. (A) Schematic of genomic region 32C4-D1 on arm 2L. Left is distal. The positions of restriction sites are shown above the line (B, BamHI; X, XbaI; Xh, XhoI). Arrows indicate start positions and directions of transcription. In the transcripts, the coding regions are shown by boxes. The MR-1 and MR-3 genomic fragments (Schmidt et al., 1999) rescue aub mutant defects. (B) Molecular defects of aub mutant alleles, with the affected amino acids indicated. The frameshift in aubN11 is caused by a 154 bp deletion which is predicted to add 16 novel amino acids after E740. Two mutants, aubK86 and aubN11, share an additional change relative to the wild-type sequence (GenBank Accession Number, X94613), 5549 G to A, which does not affect the protein sequence. Both alleles came from a single mutagenesis (Wilson et al., 1996), and this change presumably represents a polymorphism in the common parental chromosome. The third mutant, aubHN2, which was obtained in a separate screen using a different parental chromosome (Schüpbach and Wieschaus, 1991) includes three other changes, all of which are 3' to the stop codon mutation: 7304 A to C, 7326 T to A, and 7337 T to C. (C) A Western blot of ovary extracts. A band of approximately 105kDa is recognized in ovary extracts from flies wild-type at the aub locus (w1118), but not from transheterozygotes for the aubN11 and aubHN2 alleles.

View larger version (54K): [in a new window]   Fig. 2. aub mRNA and GFP-Aub expression pattern during oogenesis. (A) In situ hybridization for aub mRNA in a stage 10 egg chamber, showing expression in the nurse cells and oocyte but no localization to the posterior ooplasm. (B) A stage 8 egg chamber displaying cytoplasmic GFP-Aub in the nurse cells, identifiable by their large unstained nuclei and oocyte (arrow). Note that the nurse cell nuclei are closely surrounded by concentrated GFP-Aub (see also Fig. 5). Levels of GFP-Aub are commonly unequal in different nurse cells, as seen here. (C,D) GFP-Aub (green) and Osk (red) at the posterior pole of the oocyte in a stage 10 egg chamber. (E) Merged image of green and red channels showing colocalization (yellow).

View larger version (25K): [in a new window]   Fig. 3. Comparison of GFP-Aub and Osk protein distributions in embryogenesis. Each column shows GFP-Aub (A-C), Osk (D-F) and overlap (G-I) at the posterior pole of embryos of increasing age. (A,D,G) Embryo before pole bud formation. GFP-Aub and Osk are found in overlapping sets of particles. Some particles are labeled uniquely with GFP-Aub or Osk, but most show colocalization. (B,E,H) Embryo with pole buds forming. Both GFP-Aub and Osk are cytoplasmic, and continue to colocalize. (C,F,I) Pole cells in a precellularization embryo. GFP-Aub remains cytoplasmic, but some Osk now appears in the nuclei. Within the cytoplasm GFP-Aub and Osk are colocalized in particles of increased size. These particles are frequently concentrated on opposing faces of the nuclei, as indicated by arrowheads. All aspects of Osk protein localization are virtually identical to that displayed by a Vas-GFP fusion protein, with both proteins colocalizing in particles at all stages shown, as well as at later stages of embryogenesis (not shown).

View larger version (70K): [in a new window]   Fig. 4. Aub, Osk and Vas in structures characteristic of polar granule material. Donut-shaped structures in pole cells of cellularized embryos contain GFP-Aub (A), Osk (B,E) and Vas-GFP (D). Each of the structures containing GFP-Aub also contains Osk, as shown in the overlap of the two signals (C). Note that GFP-Aub is limited to the cytoplasm (filled arrowheads), with the nuclear Osk-containing donut-shaped structures lacking Aub (open arrowheads). All Osk-containing structures also include Vas-GFP, as seen in the overlap of the Osk and Vas-GFP signals (F). Scale bar: 5 µm. Most of the donut-shaped structures have diameters of approximately 0.5 to 1.0 µm, consistent with a diameter of 0.75 µm to 1.0 µm for polar granules and nuclear bodies as reported by Mahowald (Mahowald et al., 1976).

View larger version (48K): [in a new window]   Fig. 5. Differential effects of stau and vas mutants on GFP-Aub localization. (A) Posterior portion of a stage 10 stau mutant oocyte. GFP-Aub localization at the posterior pole is virtually eliminated. (B) stau mutant nurse cell. The appearance of GFP-Aub in perinuclear particles is unaffected. (C) vas mutant nurse cell. The perinuclear clustering of GFP-Aub particles is largely abolished. Occasionally, one or two particles may be seen. (D-E) vas heterozygous stage 10 oocyte. Both GFP-Aub (D) and Osk (E) are concentrated at the posterior pole as in wild-type. (F-G) vas mutant late stage 10 oocyte. GFP-Aub (F) localization to the posterior pole is dramatically reduced, while the level of posteriorly localized Osk (G) remains essentially normal. Although this result differs from published reports of a substantial decrease in the level of Osk protein in vas mutant ovaries, the earlier studies compared average protein levels throughout the ovary and did not differentiate between different stages of oogenesis (see text).

View larger version (79K): [in a new window]   Fig. 6. aub is required for normal pole cell formation. Embryos produced by aub heterozygous (A-D) or aub mutant (E-H) females expressing an osk-bcd transgene. (A-C,E-G) Projections of multiple z-sections showing the location of Osk (fluorescence). (A-C) Transgenic Osk protein can be found at the anterior (A) in addition to the endogenous Osk at the posterior pole (B) of precellularized embryos. After cellularization, Osk can be found in pole cells at the anterior (C) and posterior (data not shown). (D) Pole cells (arrowheads) can be identified by their round cell bodies and nuclei while those of other cells at the surface of the blastoderm have become elongated. (E,F) In early stage embryos maternally mutant for aub, only the transgenic, anterior Osk (E) accumulates to high levels. Posterior Osk (F) is absent or greatly reduced. Note that anterior localization of nos mRNA in such early stage embryos is defective, despite the anterior accumulation of Osk protein (Wilson et al., 1996). Thus, the nos mRNA localization defect cannot be attributed to the subsequent dispersal of Osk protein seen in G. (G) By the time of cellularization in embryos from aub mutant females, local concentrations of Osk at the anterior are greatly diminished, and Osk often appears dispersed from the anterior pole. Osk occasionally is found in cells along the cortex, but only rarely do these have the morphological characteristics of pole cells. Osk protein is not maintained in these cells, as we never see Osk in late embryos from aub mutants, while it persists in both ectopic and native pole cells in embryos from aub heterozygotes (not shown). (H) No obvious pole cells are found at the anterior of an embryo maternally mutant for aub.

View larger version (50K): [in a new window]   Fig. 7. GFP-Aub localization is unlikely to be required for its role in translation. (A,B) Stage 8/9 egg chamber. Only a low level of GFP-Aub (A) is localized at the posterior pole of the oocyte (arrow), even though significant Osk protein (B) has already been translated. Note that the initial appearance of GFP-Aub at the posterior pole is not delayed by a time lag in acquiring fluorescence, as the aub mRNA is not itself localized and thus the protein is not synthesized at the posterior but is likely transported from existing pools elsewhere in the egg chamber. (C) Stage 9 egg chamber, with GFP-Aub concentrated at posterior of oocyte (right). Although Grk protein (not shown) is produced in the anterodorsal corner near the oocyte nucleus (arrow) at this stage, no localization of GFP-Aub is evident. The focal plane was chosen to emphasize the nucleus; the highest concentration of GFP-Aub at the posterior lies in a different plane. (D,E) Early embryos. Compared with control embryos produced by vas heterozygous females (D), little or no Osk protein remains at the posterior pole (right) of embryos from vas mutant females (E). No difference in Osk levels is detected in stage 10 oocytes (see Fig. 4), indicating that the defect of vas mutants in translation of Osk begins only late in oogenesis.

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