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First published online 19 December 2007
doi: 10.1242/dev.016253

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Lgl and its phosphorylation by aPKC regulate oocyte polarity formation in Drosophila

Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA.

View larger version (67K): [in this window] [in a new window]   Fig. 1. lgl germline clones affect oocyte polarity. (A,B) Stau was localized at the posterior in the wild type (A) but is mislocalized in lgl4 mutant germline clones (B). (C,D) Grk and the oocyte nucleus (arrow) were localized to the anterior corner (C) but were mislocalized to a lateral position in some lgl4 mutant germline clones (D). (E,F) osk mRNA in wild-type egg chambers showed a posterior localization at stage 10 (E), whereas lgl4 germline clones displayed clear mislocalization of osk mRNA to the center of the oocyte (F). (G,H) bcd mRNA in wild-type egg chambers showed disc-shaped localization in the oocyte anterior (G, arrow), but in lgl4 germline clones bcd sometimes accumulated as dots at the anterior of the oocyte (H, arrow). (I,J) Kin:β-Gal showed posterior localization in the wild type (I) but was mislocalized to the center of the oocyte in lgl4 germline clones (J). (K,L) -Tubulin showed an anterior-to-posterior gradient in the wild-type oocyte (K) but was uniformly distributed in lgl4 germline clones (l). Mutant germline clones were marked by the absence of GFP (green) in the nuclei of the nurse cells (B,D,J) or were generated by the dominant female sterile method (F,H,L). Anterior is towards the left and posterior is towards the right in all panels.

View larger version (60K): [in this window] [in a new window]   Fig. 2 . Follicle-cell fate markers are unaffected in lgl germline clones. (A-H') Cut is an immature cell fate marker that is downregulated by Notch signaling at stage 6. Hindsight (Hnt) is expressed after stage 6 and Eyes absent (Eya) is normally expressed in anterior follicle cells and immature follicle cells. Pointed-lacZ is a posterior follicle-cell fate marker. No differences in expression of these markers between lgl4 germline clones (B,D,F,H) and the wild type (A,C,E,G) were detected. Mutant germline clones were marked by the absence of GFP (green) in the nuclei of the nurse cells.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Lgl phosphorylation can regulate oocyte polarity. Stau (A-C), osk (D-F), the quantification of Stau and osk mRNA localization (G-I), Grk (J-L), Kin:β-Gal (M-O), -Tubulin (P-R) in the wild type (driver only) (A,D,G,J,M,P), Lgl-overexpressing (B,E,H,K,N,Q) and Lgl-3A-overexpressing (C,F,I,L,O,R) egg chambers are shown. In egg chambers with Lgl overexpression, Stau (B,H), osk (E,H) and Kin:β-gal (N) were partially mislocalized, but Grk (K) showed normal localization, and -Tubulin (Q) displayed a weaker anterior-posterior gradient than in the wild type (P). In egg chambers with Lgl-3A overexpression, Stau (C,I), osk (F,I) and Kin:β-gal (O) were frequently mislocalized to the center of the oocyte, Grk and the oocyte nucleus (L) were mislocalized in some egg chambers, and -Tubulin showed higher concentrations along the cortex and lower concentration at the center of the oocyte (R).

View larger version (39K): [in this window] [in a new window]   Fig. 4. The genetic interaction between Lgl and aPKC shows that Lgl phosphorylation by aPKC can regulate oocyte polarity. (A,B) Stau was normally localized in the apkck06403 heterozygous mutant (A), but mislocalized in apkck06403 mutant germline clones (B); mutant clones were marked by the absence of nuclear GFP (green). (C,D) Loss of one copy of apkc exaggerated the Stau mislocalization phenotype stemming from Lgl overexpression. Stau localization in these backgrounds was quantified (D). (E) Stau is normally localized in aPKC-GFP overexpression. (F) Overexpression of aPKC-GFP can rescue the Stau mislocalization phenotype caused by the Lgl overexpression. (G) Stau is mislocalized in the Lgl-overexpressing egg chambers. (H) Stau localization in these backgrounds was quantified.

View larger version (51K): [in this window] [in a new window]   Fig. 5. aPKC phosphorylation of Lgl restricts Lgl localization to the oocyte posterior. (A,B) Lgl was localized at the oocyte posterior in Lgl-overexpressing egg chambers at stage 7 (A) and stage 9/10 (B). (C,D) Lgl was localized at the oocyte posterior in Lgl-GFP-overexpressing egg chambers at stage 7 (C) and stage 9/10 (D). (E,F) Lgl was localized throughout the cortex in Lgl-3A-overexpressing egg chambers at stage 7 (E) and stage 9/10 (F). (G,H) Lgl was localized throughout the cortex in Lgl-3A-GFP-overexpressing egg chambers at stage 7 (G) and stage 9/10 (H). (I,J) When Lgl or Lgl-GFP was co-overexpressed with aPKCN-GFP (GFP is not shown here) or aPKCN, Lgl or Lgl-GFP was excluded from the cortex.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Lgl interacts with Par-1 and is required for Par-1 localization. (A,B) Both the GFP-Par-1(N1S) (A) and GFP-Par-1(N1S) K* (B) were enriched at the posterior at stage 10. (C,C') The GFP-Par-1(N1S) K* lost the posterior enrichment in lgl4 germline cells. Mutant germline clones were marked by the absence of β-gal staining (red) in the nuclei of the nurse cells. (D-D") GFP-Par-1(N1S) colocalized with Lgl and was detected throughout the oocyte cortex in egg chambers with co-overexpression of Lgl-3A and GFP-Par-1(N1S). Arrows indicate the strong GFP-Par-1 and arrowheads indicate the weak GFP-Par-1. (E) Immunoblots of anti-Lgl or anti-GFP immunoprecipitation from ovaries with GFP-Par-1 (N1S) overexpression (mat-Gal4 driver) or the wild-type control (mat-Gal4 driver only). aPKC and GFP-Par-1 (N1S) proteins were precipitated by the anti-Lgl antibody from GFP-Par-1 (N1S) overexpression, and aPKC and Lgl proteins were precipitated by an anti-GFP antibody from GFP-Par-1 (N1S) overexpression. Input was one-fifth of the total ovarian extract. In anti-Lgl immunoprecipitates, controls were beads (without added antibody) and peptide blocks (+pep); in anti-GFP immunoprecipitates, controls were beads (without added antibody) and ovarian extract from wild-type flies.

View larger version (56K): [in this window] [in a new window]   Fig. 7. Lgl regulates actin organization in the oocyte. (A) GFP-Par-1 (N1S) was tightly associated with the cortical actin at the oocyte posterior. (B) In Lgl-GFP-overexpressing egg chambers, Lgl-GFP was enriched at the oocyte posterior and showed tight association with cortical actin, which had projections at the posterior of the oocyte. (C) Actin staining in the wild-type egg chamber. The weak long actin projections were indicated by arrows. (D) The long actin-rich projections (arrows) were very obvious along the oocyte cortex in Lgl-3A-overexpressing egg chambers. (E) In lgl4 germline clones, no cortical actin projections were detected. All of them are stage 10 egg chambers.

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