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First published online 15 March 2006
doi: 10.1242/dev.02319

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spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

¹ Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University, Beer-Sheva 84105, Israel.
² Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

View larger version (80K): [in a new window]   Fig. 1. DV and AP patterning defects in spn-F mutants. (A,B) Eggshells from spn-F mutant females. (A) Wild-type eggshell; (B) strongly ventralized egg shell. (C,D) Wild type embryo (C); bicaudal embryo in egg of spn-F mutant (D). Only a few percent of spn-F eggs (1-3%, depending on background) gave rise to bicaudal embryos. (E,F) Oskar protein localization at the posterior pole of stage 10 wild-type (E) and spn-F (F) egg chambers (Oskar, red; cortical Actin, green). The Oskar protein is not tightly localized to the posterior pole in the mutant. (G,H) grk RNA in situ localization in stage 9 wild-type (G) and spn-F (H) egg chambers. In most mutant egg chambers at stages 9-10, grk RNA forms a broad, fuzzy band around the anterior. (I,J) Grk protein expression in stage 9 wild-type (I) and spn-F (J) egg chambers, with Grk in red, cortical actin detected with Phalloidin in green and DNA in blue. Grk protein is strongly reduced in the mutant egg chambers. (K,L) Defects in oocyte nuclear morphology in spn-F (DNA, blue). In wild-type egg chambers (K), the DNA in the oocyte is condensed into a tight sphere. In spn-F egg chambers (L), the DNA appears fragmented.

View larger version (100K): [in a new window]   Fig. 2. Scanning electron micrographs of wild-type and spn-F mutant thorax bristles. (A-D) Wild-type bristles (A) are long and characterized by a thin and pointy tip, whereas spn-F mutant bristles (B-D) show thickening and branching, and are shorter than wild-type bristles.

View larger version (97K): [in a new window]   Fig. 3. Actin cytoskeleton organization and -tubulin staining and Tau-GFP localization in wild-type and spn-F ovaries. (A,C,E,G,I) Wild-type egg chambers; (B,D,F,H,J) spn-F egg chambers. (A,B) Young egg chambers. -tubulin, red; DNA, blue. (C,D) Stage 9 egg chambers. -tubulin, red. Abnormal aggregates of tubulin are seen at the nuclear periphery in the mutant egg chambers (arrow in D). (E,F) Abnormal accumulation of Tau-GFP is visible, associated with the mutant oocyte nucleus. Tau-GFP, green; DNA, blue; nuclear membrane, red. (G,H) Tau-GFP forms a gradient from anterior to posterior at stage 9 in both wild type and mutant; however, an abnormal accumulation of Tau-GFP occurs at the oocyte nuclear membrane in the mutant. Tau-GFP, green; DNA, blue. (I,J) Abnormal clumps of actin can be seen in the mutant egg chamber (J), compared with wild type (I). Rhodamine-phalloidin staining, red; DNA, blue.

View larger version (106K): [in a new window]   Fig. 4. kin:ß-Gal and Nod:ß-Gal localization in wild-type and spn-F egg chambers. (A,C,E) Wild-type egg chambers; (B,D,F) spn-F egg chambers; (C,D) stage 6 egg chambers; (E,F) stage 8 egg chambers. (A,B) kin:ß-Gal localization (green). The localization to the posterior pole appears normal in the mutant egg chambers. (C-F) Nod:ß-Gal localization (green). The localization of Nod:ß-Gal appears less pronounced in the posterior in the stage 6 mutant egg chamber, and less concentrated around the oocyte nucleus in the stage 8 mutant than in wild type.

View larger version (72K): [in a new window]   Fig. 5. bcd and encore RNA distribution in wild-type and spn-F ovaries. (A-D) bcd (A,B) and encore (C,D) mRNA in wild-type (A,C) and spn-F (B,D) egg chambers. The localization of these anterior RNAs appears normal in the mutant egg chambers.

View larger version (73K): [in a new window]   Fig. 6. Intracellular localization of Spn-F protein in Drosophila ovaries and its association with Dynein light chain in vitro. (A-D) Spn-F protein (red) accumulates at the posterior pole in egg chambers until stage 6 (A), and accumulates at the anterior cortex after stage 8 (B, stage 9 egg chamber) and also in a punctate pattern in the nurse cells. (C) In a colcemid-treated egg chamber, Spn-F protein does not localize to the anterior cortex and a more punctate distribution of Spn-F is detected in the nurse cells. (D) In egg chambers from Dynein heavy chain mutant flies, Spn-F protein does not localize significantly to the anterior cortex and a more punctate distribution in of Spn-F is detected in the nurse cells. (E) GST pull-down assay for the in vitro binding of His-Spn-F to GST-Ddlc-1. GST-Ddlc1 and Spn-F were mixed with glutathione-sepharose 4B resins. As a negative control, GST-conjugated resins were incubated with Spn-F. (Lanes 1-3) Input proteins: GST (lane 1), GST-Ddlc (lane 2) and His-Spn-F (lane 3). (Lane 4) GST-Ddlc and Spn-F; (lane 5) GST and Spn-F. When Ddlc-GST was pulled down, Spn-F was also precipitated (lane 4), whereas this was not the case when GST alone was pulled down (lane 5). (F) In vitro binding of Spn-F to GST-Ddlc. Extracts of wild-type ovaries were incubated with GST-Ddlc-1 or with GST alone, GST was pulled down, and the eluate was electrophoresed and probed with an anti-Spn-F antibody. (Lane 1) Control: GST-conjugated resins and ovarian extract. (Lane 2) GST-Ddlc and an ovarian extract. Spn-F associates directly with Dynein light chain in vitro. (Lane 3) Control: ovarian extract only.