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First published online 5 January 2005
doi: 10.1242/dev.01590

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Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

Matthieu Cavey^*, Sirine Hijal, Xiaolan Zhang and Beat Suter

Department of Biology, McGill University, 1205 Dr Penfield Avenue, Montréal, QC, H3A 1B1, Canada

View larger version (21K): [in a new window]   Fig. 1. vls genomic region. (A) Four transcription units are found in a 12 kb stretch of DNA in region 38B (Butler et al., 2001). Df(2L)be408 uncovers CG10728, chk2 and a small region of the barr 5' end. Df(2L)pr2b deletes 38B1-2 to 38D2-E1. The fragments indicated as P[]were reintroduced by P-element-mediated transformation and recombined onto the Df(2L)pr2b or Df(2L)be408 chromosomes. chk2null flies (Df(2L)be408, P[w+ CG10728+/vls+] / Df(2L)pr2b, P[w+ barren+]) are viable and fertile, demonstrating that chk2 does not have any essential function for development. E, EcoRI; B, BamHI; H, HindIII. (B) Magnification of the vls gene drawn to scale. Exons are shown as dark boxes, introns and untranslated regions (UTRs) as thin lines. The position of premature stop codons in the three vlsEMS alleles is indicated with the corresponding nucleotide substitution and codon change. The predicted 3' UTRs of vls and chk2 overlap over 127 bp. The asterisk indicates the N-terminal position of the polypeptide used for antibody production.

View larger version (26K): [in a new window]   Fig. 2. chk2 is translationally repressed by orb and vls during oogenesis. The indicated ovary extracts were probed with -Chk2 (Masrouha et al., 2003) and -eIF4A antibodies as a loading control. Protein levels were quantified using a phosphorimager. The upper band in OreR and chk2null mutants is crossreacting material of unknown identity. Chk2 levels were normalized to eIF4A levels and expressed as a percentage of normalized Chk2 levels in wild type (OreR), which is arbitrarily set at 100%.

View larger version (51K): [in a new window]   Fig. 3. Drosophila Vls resembles Homo sapiens methylosome protein 50 (MEP50). The alignment of the two predicted protein sequences was made using ALIGN Query (GENESTREAM SEARCH network server IGH Montpellier, France). Identical residues are highlighted in black and conserved residues in gray. The WD domains of MEP50 (Friesen et al., 2002) are boxed and numbered in blue, the predicted WD domains of Vls (PROSITE) in green.

View larger version (58K): [in a new window]   Fig. 4. Western analysis of Vls. Ponceau Red staining serves as a loading control and is shown below the corresponding blot. (A) Anti-Vls antibodies detect a protein of 42 kDa in wild-type ovaries and in ovaries expressing one copy of the CG10728+/vls+ rescue construct. This antigen is not detected in vlsnull mutants, nor in vlsEMS hemizygotes and trans-heterozygotes. Genotypes: vlsnull - Df(2L)be408 / Df(2L)pr2b, P[w+ barr+]; vlsnull vls+ - Df(2Lbe408, P[w+ CG10728+/vls+] / Df(2L)pr2b, P[w+ barr+]; and Df - Df(2L)be408. (B) Developmental expression profile of Vls. vlsnull ovary extracts serve as a control for the specificity of the -Vls antibodies. Other protein extracts were collected from wild-type flies. The faint low molecular weight band does not appear to originate from ovaries or embryos.

View larger version (94K): [in a new window]   Fig. 5. Distribution of Vls-eGFP in ovaries and embryos. Transgenic flies express two copies of vls-eGFP. Anterior is towards the left, posterior towards the right. Vls-eGFP is cytoplasmic. (A) In the germarium, Vls-eGFP signal is found predominantly in the germline and at lower levels also in the somatic follicle cells, and occasionally accumulates in aggregates up to stage 1 egg chambers. Vls-eGFP is uniform in the nurse cells and the developing oocyte throughout the rest of oogenesis. (B) Stage 1-4 egg chambers, (C) stage 8 and (D,E) stage 10. Vls-eGFP signal is generally fairly weak in the oocyte and more difficult to detect against the autofluorescent yolk particles (D). However, the signal is still significantly higher than in control yw ovaries without vls-eGFP (E). (F,G) 0- to 1-hour-old embryos from vls-eGFP (F) and yw (G) mothers. (H,I) 1- to 2-hour-old embryos from vls-eGFP mothers. Vls-eGFP signal is detected in somatic and germ cell cytoplasm. pc, pole cells; sc, somatic cells.

View larger version (74K): [in a new window]   Fig. 6. osk mRNA and Osk protein distribution appear normal initially in vls mutant ovaries but Osk protein disappears from the posterior pole after stage 11. (A) In situ hybridization with an osk probe to a vlsnull stage 10 egg chamber. osk mRNA is correctly concentrated at the posterior of the oocyte in vlsnull mutants. (B-E'') Immunostaining with -Osk antibodies on wild-type and vls mutant ovaries. Osk signal is tightly concentrated at the posterior of stage 9-10 oocytes of wild type (B-B') and vlsnull vls+ (C-C'), as well as in vlsPG65 (D-D') and vlsnull (E-E') females. However, around stage 11, Osk signal appears slightly weaker at the posterior of vlsPG65 (D'') and significantly weaker in vlsnull oocytes (E''). This defect is even more pronounced in vlsnull after stage 11, where usually no posterior Osk signal is detected (not shown).

View larger version (50K): [in a new window]   Fig. 7. vls is required for normal accumulation of Osk isoforms. Western blots of the indicated ovary extracts probed with -Osk antibodies. Loading of approximately equal amounts of proteins shows that vlsnull ovaries contain only very little Osk compared with wild type (OreR; left blot). -Osk antibodies recognize the Long isoform of Osk, as well as the hyperphosphorylated (57 kDa) and hypophosphorylated (55 kDa) forms of Short Osk (arrows). For the blot on the right, about ten times as much protein extracts were loaded onto the vlsnull lane compared with the loading for the OreR lane. Ponceau staining of the membrane (Ponc., left blot) and reprobing of the blot with -Tubulin antibodies (right blot) were used as loading controls.

View larger version (57K): [in a new window]   Fig. 8. Posterior Vas localization requires vls. Wild type has two copies of vas-eGFP. vlsnull vls+, vlsPG65 and vlsnull have one copy of vas-eGFP. Fixed ovaries are shown here. Live ovaries show a similar pattern although nuage signal is generally stronger than in fixed ovaries. (A-D) Stage 1-5 egg chambers, (A'-D') stage 10 egg chambers. (A,A') Wild-type localization of Vas-eGFP to nuage and to the posterior of the oocyte is observed in Sp/SM1 background. Nuage localization in vls mutants appears normal initially (C,D) and slightly reduced in stage 10 egg chambers (C',D'); however, we did not observe this reduction in live ovaries (data not shown). Posterior localization of Vas-eGFP in the oocyte is undetectable in vlsnull mutants (D') and dramatically reduced in vlsPG65 hemizygotes (C'). This defect is rescued by the introduction of the vls+ transgene (B'). The levels of posterior Vas-eGFP appear reduced in vlsnull vls+ oocytes, most probably because of the lower copy number of vas-eGFP. (E-H') Vas-eGFP is not detected at the posterior of young embryos from vls mutant mothers. (E-H) 0- to 1-hour-old embryos, (E'-H') 2- to 3-hour-old embryos. Vas-eGFP accumulates at the posterior of embryos (E,F) and then inside newly formed pole cells (E',F') in wild-type and vlsnull vls+ background, but not in embryos from vlsPG65 hemizygous (G,G') and vlsnull mutant (H,H') mothers.