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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00630

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The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP-Staufen

Sophie G. Martin*,{dagger}, Vincent Leclerc*,{ddagger}, Katie Smith-Litière§ and Daniel St Johnston

The Wellcome Trust/Cancer Research UK Institute and the Department of Genetics, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QR, UK



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  		Fig. 2. Crossing scheme for isolating mutants defective in GFP-Staufen localisation. The asterisk indicates the mutagenised chromosome. The red X shows a mitotic recombination event between the two FRT sequences, which occurs after induction of FLP expression in response to heat-shock. ovoD1 is a dominant female-sterile mutation that blocks oogenesis at early stages. Within a clonal ovary, the only germline cysts that develop to later stages are those that are homozygous for the mutagenised chromosome.

 


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  		Fig. 7. orb and spn-E are required for the organisation of the microtubule cytoskeleton at stage 9. (A-D) {alpha}-tubulin staining. (E-H) Merge of eight consecutive frames of a time-lapse movie of autofluorescent particles. A static particle shows as a dot, whereas a moving particle forms a line. Wild-type oocytes (A,E) show a characteristic anterior to posterior gradient of microtubules and very little cytoplasmic movement. By contrast, spn-E4E2-14/spn-Ehls{Delta}157 (B,F) and orb7E4-5 (C,G) oocytes display thick microtubule bundles and rapid circular cytoplasmic movements. Similar observations were made in spn-E2A9-14 and spn-E8D4-11, and in all orb alleles described in panel I. (D,H) Wild-type patterns of microtubule distribution and cytoplasmic streaming are restored in half of orb yps double-mutant oocytes (ypsJM2 orbmel/ypsJM2 orbF303). (I) Allelic series of orb alleles. The alleles mentioned in the table were crossed to orbF343 and classified according to the phenotype of transheterozygous females. The ventralised eggs laid by these females were unfertilised. `early arrest' indicates that egg chambers in these females arrested development during early oogenesis and failed to reach vitellogenic stages. (J) Western blot analysis of Oskar and ORB in orb, orb yps and spn-E ovaries. {alpha}-Tubulin (TUB) was used as a loading control. Longer exposure (on the right) shows the presence of low levels of ORB in spn-E mutant ovaries. Similar observations were made with spn-E2A9-14, spn-E4E2-14 and spn-E8D4-11 alleles. Exact genotypes are as follows: TM3, ypsJM2 orbF303/TM3; yps, ypsJM2/ypsJM2 orbF303; orb, orbmel/ypsJM2 orbF303; orb yps, ypsJM2 orbmel/ypsJM2 orbF303; spn-E/TM3, spn- Ehls{Delta}157/TM3; spn-E, spn-E4E2-14/spn-Ehls{Delta}157.

 


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  		Fig. 1. Localisation of GFP-Staufen. (A-C) Expression of GFP-Staufen (green) in fixed egg chambers counterstained with rhodamine-phalloidin, which labels F-actin (red). (A) Stage 6 egg chamber, showing the accumulation of GFP-Staufen in the oocyte. (B) Stage 9 egg chamber, showing the localisation of GFP-Staufen to the posterior of the oocyte. (C) Stage 10b egg chamber, in which GFP-Staufen localises both to the anterior and the posterior pole of the oocyte. (D-F) Localisation of GFP-Staufen in stage 9 egg chambers. (D) Wild type. (E) btz2. (F) grk2B6/grk2E12. (G,H) Localisation of GFP-Staufen in stage 11 egg chambers. (G) Wild type. (H) exuVL/exuQR. Note that the posterior localisation of GFP-Staufen is unaffected. Anterior is to the left, posterior to the right in this and all subsequent Figures.

 


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  		Fig. 3. Classes of mutants affecting the localisation of oskar mRNA. GFP-Staufen (green) and rhodamine-phalloidin staining (red) are shown in the left panels (A,C,E,G,I,K). oskar in situ hybridisations are shown on the right (B,D,F,H,J,L). (A,B) Wild-type egg chambers. (C,D) vagabond7A1-3. In this mutant, GFP-Staufen and oskar mRNA fail to localise to the posterior and oskar mRNA accumulates at the anterior of stage 9 oocytes. In C, the oocyte nucleus is also mislocalised to the lateral oocyte cortex (arrow). (E,F) fraenkel4B2-7, a member of the class of mutants in which GFP-Staufen and oskar mRNA localise ectopically to the centre of the oocyte. (G-J) 2B1-8 egg chambers. In this mutant, GFP-Staufen and oskar mRNA localise to the posterior of the oocyte in stage 9 (G), but fail to be maintained there in later stages (I,J). In the stage 10A oocyte shown in H, oskar mRNA seems to be falling off from the posterior cortex. (K,L) glissade9E9-3 exemplifies the class of mutants in which GFP-Staufen and oskar mRNA localise to the posterior part of the oocyte, but not in a cortical crescent.

 


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  		Fig. 4. Classes of mutants affecting the localisation of bicoid mRNA. GFP-Staufen (green) and rhodamine-phalloidin staining (red) are shown in the left panels (A,C,E). bicoid in situ hybridisations are shown on the right (B,D,F). (A,B) Wild-type egg chambers. (C,D) 7D8-15 egg chambers, an example of the class of mutants in which bicoid mRNA fails to concentrate along the anterior cortex in early stages of oogenesis. (E,F) larsen2B6-3, a member of the second class of bicoid-specific mutants, in which bicoid mRNA localisation in early stages is indistinguishable from wild type, but GFP-Staufen fails to concentrate at the anterior cortex of stage 12 oocytes.

 


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  		Fig. 5. Classes of mutants affecting the localisation of both oskar and bicoid mRNA. In situ hybridisations for bicoid are shown on the left (A,C,E,G) and hybridisations for oskar are shown on the right (B,D,F,H). (A,B) Wild-type egg chambers. (C,D) boussole8F8-6. This mutant shows ectopic localisation of oskar mRNA to the centre of the oocyte, and posterior accumulation of bicoid mRNA in a subset of oocytes. (E,F) abruzzi7C3-10 is a member of a large class of mutants, in which the oocyte nucleus is frequently mislocalised (see arrow in panel E) and bicoid mRNA associates with the cortex proximal to the nucleus. In these mutants, oskar mRNA also fails to adopt a wild-type localisation pattern. (G,H) mertz8077, an example of the class of mutants in which both oskar and bicoid mRNAs fail to localise to their respective poles and are diffuse in the oocyte.

 


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  		Fig. 6. Other phenotypes of mutants found in the screen. (A) sorcière4E5-2. In this mutant, the bulk of GFP-Staufen (green; top inset) is blocked in the nurse cells and co-localises with the ring canals stained with rhodamine-phalloidin (red; bottom inset). GFP-Staufen also fails to accumulate to the anterior of the oocyte in later stages. (B) trou9E4-15 shows a defect of adhesion between the posterior follicle cells and the oocyte. This mutant is also defective in bicoid and oskar mRNA localisation. (C) In sedov9E9-3, a member of the class of mutants in which oskar mRNA is diffusely localised in the posterior part of the oocyte, the organisation of the follicle cells is aberrant. This phenotype may reflect a premature migration of `centripetal' follicle cells between the oocyte and the nurse cells. Alternatively, it may indicate an overproliferation of follicle cells. A similar phenotype was observed in wellman and vagabond mutants. (D) nain4A3-6 is a member of the class of mutants in which the oocyte fails to acquire wild-type size. The rare oocyte escapers that grow fail to localise GFP-Staufen in a wild-type manner. (E) 4B7-11, one of the two mutants in which the egg chamber contains more than 16 germ cells.

 

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© The Company of Biologists Ltd 2003