Bazooka regulates microtubule organization and spatial restriction of germ plasm assembly in the Drosophila oocyte

Abstract

Localization of the germ plasm to the posterior of the Drosophila oocyte is required for anteroposterior patterning and germ cell development during embryogenesis. While mechanisms governing the localization of individual germ plasm components have been elucidated, the process by which germ plasm assembly is restricted to the posterior pole is poorly understood. In this study, we identify a novel allele of bazooka (baz), the Drosophila homolog of Par-3, which has allowed the analysis of baz function throughout oogenesis. We demonstrate that baz is required for spatial restriction of the germ plasm and axis patterning, and we uncover multiple requirements for baz in regulating the organization of the oocyte microtubule cytoskeleton. Our results suggest that distinct cortical domains established by Par proteins polarize the oocyte through differential effects on microtubule organization. We further show that microtubule plus-end enrichment is sufficient to drive germ plasm assembly even at a distance from the oocyte cortex, suggesting that control of microtubule organization is critical not only for the localization of germ plasm components to the posterior of the oocyte but also for the restriction of germ plasm assembly to the posterior pole.

Fig. 1

Characterization of the baz^X-82 allele. (A) Domain structure of Baz. Asterisks denote inhibitory (red) and activating (green) regulatory phosphorylation sites. The position of the X-82 stop codon is indicated by a dashed line. Numbers represent amino acid position. (B) Anti-Baz immunoblot of total protein from wild-type and baz^X-82 ovaries. Tubulin serves as a loading control. A truncated protein is detected in baz^X-82 lysates.

Fig. 2

baz^X-82 mutant embryos display ectopic germ plasm localization. (A-D) In situ hybridization to nos (A,B) and osk (C,D) mRNAs in early wild-type (A,C) or baz^X-82 (B,D) embryos. nos and osk show abberrant localization in baz^X-82 embryos. (E,F) Confocal images of Vas immunofluorescence in early wild-type (E) or baz^X-82 (F) embryos. (G,H) In situ hybridization to bcd mRNA in early wild-type (G) or baz^X-82 (H) embryos. bcd mRNA localization is not disrupted by baz^X-82. (I,J) Double fluorescent in situ hybridization (FISH) to osk (green) and nos (red) mRNAs in early wild-type (I) or baz^X-82 (J) embryos.Merged confocal images (top panels) show that osk and nos mRNAs colocalize in ectopic patches in baz^X-82 embryos. Individual channels are shown below. (K, L) In situ hybridization to nos mRNA in pre-blastoderm wild-type (K) and baz^X-82 (L) embryos.

Fig. 3

baz^X-82 affects germ plasm localization during oogenesis. Confocal images of wild-type (WT) (A,C,E,G,I) and baz^X-82 (B,D,F,H,J) egg chambers. (A-D) FISH to osk mRNA in stage 10 oocytes. (E-J) Direct visualization of Vas-GFP (E,F) in stage 10 oocytes (arrow in F) and Osk-GFP (G,H) and Vas-GFP (I,J) in stage 14 oocytes. Germ plasm mRNA (osk) and protein (Osk, Vas) components are ectopically localized in baz^X-82 oocytes. (K,L) Confocal images of egg chambers expressing Osk-GFP. (K) Wild-type egg chamber. (L) A rare baz⁴ egg chamber where an oocyte is specified. Nuclei are labeled with WGA.

Fig. 4

Microtubule organization and polarity is disrupted in baz^X-82 oocytes. Confocal images of wild-type (A,C,E,G,I,K) and baz^X-82 (B,D,F,H,J,L) stage 9 oocytes. (A,B) Anti-α-tubulin immunofluorescence reveals an A-P gradient of microtubules in the oocyte which is lost in baz^X-82. (C-F) Anti-ßgal immunofluorescence. (C,D) Kin-ßgal accumulation is absent (not shown) or ectopic in the baz^X-82 oocyte. (E,F) Nod-ßgal accumulates at the posterior of baz^X-82 oocytes. (G, H) Grk localization follows the nucleus in baz^X-82 oocytes. (I,J) FISH to bcd mRNA. bcd is ectopically localized in baz^X-82 oocytes. (K,L) pnt-lacZ expression in posterior follicle cells is not affected by baz^X-82. Actin is detected with phalloidin (red); nuclei are labeled with WGA (blue). (M,N) Merge of sixteen consecutive frames of a confocal time-lapse movie of autofluorescent yolk granules to visualize ooplasmic streaming in wild-type (M) and baz^X-82 (N) late stage oocytes. No streaming is observed in baz^X-82 oocytes. n: oocyte nucleus.

Fig. 5

Baz^X-82 is mislocalized in the oocyte. Confocal images of (A) Baz-GFP and (B) Baz^X-82-GFP in stage 9 oocytes. Baz-GFP is localized to the anterior and lateral oocyte cortex, but is depleted at the posterior (arrowheads). Baz^X-82-GFP is diffuse in the cytoplasm in addition to uniform weak cortical accumulation. Actin is labeled with phalloidin (red) and nuclei with WGA (blue). GFP channel shown separately on the right.

Fig. 6

Par-1 is mislocalized in baz^X-82 oocytes. (A-F) Confocal images of wild-type (A,C,E) and baz^X-82 (B,D,F) stage 10 egg chambers. (A,B) Anti-Par-1 immunostaining. Posterior enrichment of Par-1 is absent in baz^X-82 oocytes (compare regions indicated by arrows). (C-F) Double immunostaining for Par-1 (green) and Osk (blue). Par-1 colocalizes with ectopic Osk in baz^X-82 oocytes (arrowheads). (G-H) Higher power confocal images of the follicle cell epithelium of wild-type (G,I) and baz^X-82 egg chambers (H,J) immunostained with anti-Baz (G,H) or anti-Par-1 (I,J) antibodies. Nuclei are labeled with WGA (blue). The anti-Baz antibody detects both wild-type Baz and Baz^X-82 in the follicle cells but is not adequate for detection of either in the oocyte. Although Baz^X-82 is apically localized, it fails to accumulate at adherens junctions.

Fig. 7

Germ plasm assembly occurs ectopically in grk mutant egg chambers. Confocal images of wild-type (A,C,E,G) or grk^2B/HF (B,D,F,H) egg chambers. (A,B) FISH to osk mRNA in stage 9 oocytes. (C,D) Osk localization in stage 9 oocytes. (E-H) Vas-GFP localization in stage 10 (E,F) and stage 14 (G,H) oocytes. Germ plasm is ectopically assembled in grk mutant egg chambers. Actin is labeled with phalloidin (red), nuclei with WGA (blue).